RT- PCR in situ: Light and Electron Microscopy

PCR/RT-PCR in situ LIGHT and ELECTRON MICROSCOPY

Methods in Visualization Series Editor: Gérard Morel

In Situ Hybridization in Light Microscopy Gérard Morel and Annie Cavalier Visualization of Receptors: In Situ Applications of Radioligand Binding Emmanuel Moyse and Slavica M. Krantic Genome Visualization by Classic Methods in Light Microscopy Jean-Marie Exbrayat Imaging of Nucleic Acids and Quantitation in Photonic Microscopy Xavier Ronot and Yves Usson In Situ Hybridization in Electron Microscopy Gérard Morel, Annie Cavalier, and Lynda Williams PCR/RT - PCR In Situ Light and Electron Microscopy Gérard Morel and Mireille Raccurt

PCR/RT-PCR in situ LIGHT and ELECTRON MICROSCOPY Gérard Morel, Ph.D., D.Sc. Mireille Raccurt, Ph.D.

CRC PR E S S Boca Raton London New York Washington, D.C.

Library of Congress Cataloging-in-Publication Data Morel, Gérard. PCR/RT-PCR in situ : light and electron microscopy / Gérard Morel, Mireille Raccurt. p. cm. -- (Methods of visualization) Includes bibliographical references and index. ISBN 0-8493-0041-X (alk. paper) 1. Microscopy--Technique. 2. Electron microscopy--Technique. 3. Polymerase chain reaction. 4. Reverse transcriptase. I. Raccurt, Mireille. II. Title. III. Series. QH207 .M67 2002 570′.28′2--dc21

2002073649

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from CRC Press LLC for such copying. Direct all inquiries to CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe.

Visit the CRC Press Web site at www.crcpress.com © 2003 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-0041-X Library of Congress Card Number 2002073649 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper

SERIES PREFACE Visualizing molecules inside organisms, tissues, or cells continues to be an exciting challenge for cell biologists. With new discoveries in physics, chemistry, immunology, pharmacology, molecular biology, analytical methods, etc., limits and possibilities are expanded, not only for older visualizing methods (photonic and electronic microscopy), but also for more recent methods (confocal and scanning tunneling microscopy). These visualization techniques have gained so much in specificity and sensitivity that many researchers are considering expansion from in-tube to in situ experiments. The application potentials are expanding not only in pathology applications but also in more restricted applications such as tri-dimensional structural analysis or functional genomics. This series addresses the need for information in this field by presenting theoretical and technical information on a wide variety of related subjects: in situ techniques, visualization of structures, localization and interaction of molecules, and functional dynamism in vitro or in vivo. The tasks involved in developing these methods often deter researchers and students from using them. To overcome this, the techniques are presented with supporting materials such as governing principles, sample preparation, data analysis, and carefully selected protocols. Additionally, at every step we insert guidelines, comments, and pointers on ways to increase sensitivity and specificity, as well as to reduce background noise. Consistent throughout this series is an original two-column presentation with conceptual schematics, synthesizing tables, and useful comments that help the user to quickly locate protocols and identify limits of specific protocols within the parameter being investigated. The titles in this series are written by experts who provide to both newcomers and seasoned researchers a theoretical and practical approach to cellular biology and empower them with tools to develop or optimize protocols and to visualize their results. The series is useful to the experienced histologist as well as to the student confronting identification or analytical expression problems. It provides technical clues that could only be available through long-time research experience. Gérard Morel, Ph.D. Series Editor

V

ACKNOWLEDGMENTS The authors particularly thank their students Brice Ronsin, Sophie Recher, Elara Moudilou, Cécile Vivancos for invaluable help in developing the methodology. We also acknowledge Françoise de Billy for her expertise in plant biology and for the illustrations she provided. We are grateful to Professors Tomas Garcia-Caballero and Annie Cavalier for their help in this project. We also thank John Doherty for his excellent English translation. We thank the different corporations, Applied BioSystem, Hybaid, and MJ Research, for their material assistance and all the technical description provided. This work was carried out in the framework of the European “Leonardo Da vinci” project (Grant F/96/2/0958/PI/II.1.1.c/FPC), in association with Claude Bernard-Lyon 1 University.

VII

THE AUTHORS Gérard Morel, Ph.D., D.Sc., is a research director at the National Center of Scientific Research (CNRS), at University Claude Bernard-Lyon 1, Villeurbanne, France. Dr. Morel obtained his M.S. and Ph.D. degrees in 1973 and 1976, respectively, from the Department of Physiology of Claude Bernard University-Lyon 1. He was appointed an assistant of histology at the same university in 1974 and became Doctor of Science in 1980. He was appointed by CNRS in 1981 and became research director in 1989. Dr. Morel is a member of the American Endocrine Society, The International Society of Neuroendocrinology, The Society of Neuroscience, The American Society for Cell Biology, Société Française des Microscopies, Société de Biologie Cellulaire de France, and Société de Neuroendocrinologie Expérimentale. He has been the recipient of research grants from the European Community, INSERM (National Institute of Health and Medical Research), La Ligue contre le Cancer, l’ARC (Association de Recherche contre le Cancer), Claude Bernard University, and private industry. Dr. Morel’s current major research interests include the internalization and cellular trafficking of ligand and receptor molecules (in particular, nuclear receptors for peptides), the regulation of gene expression, and paracrine interactions (low gene expression level of ligand in target tissue). Mireille Raccurt, Ph.D., is an engineer in biology in a laboratory of the CNRS (Molecular Physiology), at Claude Bernard-Lyon 1 University, Villeurbanne, France. She has worked in a number of different laboratories, forming the basis for her knowledge and expertise in the fields of cellular and molecular biology. She has published more than 20 papers in the field of protein and nucleic acid detection from normal and pathological tissues. She has taught histology at the Lyon School of Medicine from 1976 to 1989. Moreover, she has regularly organized and taught the theory and practical courses of immunocytology, in situ hybridization, and PCR in training courses at light and electron microscopic levels in different European countries. She has organized six meetings or workshops. Her expertise and current research interests include the localization and regulation of hormone and receptor gene expressions, correlated with signaling molecules in normal and tumoral states. Most recently, she has become interested in extrapituitary expression of growth hormone in fetal and adult tissues and its regulation in mammary gland.

IX

CONTENTS

General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

XIII

Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

XV

Chapter 1 - General Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

Chapter 2 - Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21

Chapter 3 - Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

47

Chapter 4 - Reverse Transcription (RT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65

Chapter 5 - Polymerase Chain Reation (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . .

87

Chapter 6 - Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

119

Chapter 7 - Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

147

Chapter 8 - Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

177

Chapter 9 - Controls and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

233

Chapter 10 - Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

277

Chapter 11 - Examples of Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

323

Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

343

A - Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

349

B - Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

355

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

389

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

407

XI

General Introduction

GENERAL INTRODUCTION Over the past decade, no new procedure in molecular biology has achieved such an exceptional degree of biotechnical acceptance as the polymerase chain reaction (PCR). This in vitro enzymatic amplification of particular genetic sequences is now a basic tool in experimental work, and has become a highly standardized process. More recently, improved enzymes, automated routines, and the possibility of carrying out the reaction on DNA chips have opened entirely new applications for the method. In addition to its impact in laboratory research and medical diagnostics, PCR holds promise for significant advances in quality control for agricultural products, and has become the most powerful weapon in the arsenal of forensic science. Powerful as PCR may be, however, it still involves the destruction of cells and tissue, and morphologists whose interests are associated with intact structures have remained unsatisfied. What they needed was a way to adapt PCR methods to undamaged cells or tissue sections to detect small numbers of copies of DNA or RNA in situ, while still preserving morphology. And in 1990, Haase et al. actually succeeded in amplifying lentiviral DNA in infected cells and detecting the amplification product by in situ hybridization. And so it was that in situ PCR came into being. This demonstrates that a “mere” technical innovation is sometimes all that is needed to give a fresh impetus to research. One of the first successes achieved by the new technique was to confirm the relationship between HIV and AIDS. Initially, it had been found that only 1% of CD4 cells in the blood of asymptomatic seropositive subjects was infected by HIV, and it was difficult to see how such a small number of infectious particles could be responsible for such a serious condition. In 1992, however, several teams, including those of G. Nuovo and O. Bagasra, then that of Kominoth, began developing in situ PCR, which combined the amplifying power of the PCR with the ability of in situ hybridization to localize target sequences. Using this technique, it

was demonstrated that in reality 30 to 40% of circulating CD4 cells were infected by HIV, thus making it clear that AIDS was essentially a viral disease, and underscoring the need for direct therapy against viral replication. In situ PCR and RT-PCR provided the first means of detecting minute quantities of DNA or RNA in nondisrupted cells and tissue, with the possibility of subsequently detecting the amplified product at the site of origin. But if in situ PCR is to be successful, it requires considerable optimization—not just of the PCR cycling but also of the fixation step and tissue processing, the PCR tools and reagents used for the amplification, and the detection procedure. The objective of this book is to give scientists (morphologists, pathologists, or molecular biologists) the information they need about the basic approach to histological analysis, biochemistry of PCR, and to provide molecular biologists with a practical approach to histological analysis. Chapter 1 is a general presentation of in situ PCR/RT-PCR and the variants. The parameters for fixation, tissue processing, and enzyme digestion are set out in Chapters 2 and 3. Reverse transcription (RT) and amplification techniques are described, with theoretical considerations and practical step-by-step guidance, in Chapters 4 and 5. Detection procedures are discussed in Chapters 6 and 7. And, given that in situ PCR can be combined with electron microscopy, the basic principles of these methods are presented in detail in Chapter 8. Numerous controls are, of course, needed to check for diffusion and potential causes of background, negative, or nonspecific signals. Finally, the causes of the false positives and false negatives associated with the different techniques are outlined in Chapter 9, with recommendations on how to avoid them. Chapter 10 provides guidelines that should help experimenters work out their own

XIII

General Introduction in situ PCR / RT-PCR protocol, although in the last analysis it is the empirical conditions themselves that dictate the precise details of any given protocol. Some examples of results are illustrated in Chapter 11. Methods for preparing the different reagents are given in the Appendices.

XIV

Finally, it is the hope of the authors, who are themselves actively involved in the development of in situ PCR/RT-PCR, that the present work will provide practical solutions to some of the problems encountered by experimenters in the implementation of these complex, still-evolving techniques.

Abbreviations

ABBREVIATIONS AEC ATP BCIP bp cDNA CTP DNA DAB dATP dCTP DEPC dGDP dGMP dGTP DNase dNTP DTT dUTP EDTA Fab (Fab′)2 Fc FITC GTP Ig IgG kb kDa MM mRNA Mw NBT NTP Oligo (dT) PBS PCR PF RNA RNase rRNA

➫ 3-amino-9-ethylcarbazole ➫ adenosine triphosphate ➫ 5-bromo-4-chloro-3-indolyl phosphate ➫ base pair ➫ complementary deoxyribonucleic acid ➫ cytosine triphosphate ➫ deoxyribonucleic acid ➫ 3′-diaminobenzidine tetrahydrochloride ➫ deoxy-adenosine triphosphate ➫ deoxy-cytosine triphosphate ➫ diethyl-pyrocarbonate ➫ deoxyguanosine-5′-diphosphate ➫ deoxyguanosine-5′-monophosphate ➫ deoxyguanosine 5′-triphosphate ➫ deoxyribonuclease ➫ deoxynucleoside triphosphate ➫ dithiotreitol ➫ deoxyuridine-5′-triphosphate ➫ ethylene diamine tetraacetic acid ➫ immunoglobulin fragment obtained by proteolysis (papaine) ➫ immunoglobulin fragment obtained by proteolysis (pepsine) ➫ immunoglobulin fragment obtained by proteolysis (papaine) ➫ fluorescein isothiocyanate ➫ guanosine triphosphate ➫ immunoglobulin ➫ immunoglobulin G ➫ kilobase ➫ kilodalton ➫ molecular mass ➫ messenger ribonucleic acid ➫ molecular weight ➫ nitroblue tetrazolium ➫ nucleoside triphosphate ➫ oligo-deoxythymidine ➫ phosphate buffer saline ➫ polymerase chain reaction ➫ paraformaldehyde ➫ ribonucleic acid ➫ ribonuclease ➫ ribosomic ribonucleic acid

XV

Abbreviations rt RT RT-PCR SSC TH Tm tRNA Tw U UDP UTP v/v w/v

XVI

➫ room temperature ➫ reverse transcription ➫ reverse transcription–polymerase chain reaction ➫ standard saline citrate ➫ hybridization temperature ➫ melting temperature ➫ transfer ribonucleic acid ➫ washing temperature ➫ unit (enzymatic activity) ➫ uridine-5′-diphosphate ➫ uridine-5′-triphosphate ➫ volume/volume ➫ weight/volume

CONTENTS

General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

XIII

Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

XV

Chapter 1 - General Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

Chapter 2 - Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21

Chapter 3 - Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

47

Chapter 4 - Reverse Transcription (RT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65

Chapter 5 - Polymerase Chain Reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . .

87

Chapter 6 - Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

119

Chapter 7 - Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

147

Chapter 8 - Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

177

Chapter 9 - Controls and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

233

Chapter 10 - Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

277

Chapter 11 - Examples of Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

323

Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

343

A - Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

349

B - Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

355

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

389

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

407

XI

Chapter 1 General Principles

Contents

CONTENTS 1.1 Polymerase Chain Reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 1.1.2 1.1.3

5

Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Target Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3.1 Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3.2 Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3.3 Experimental Conditions . . . . . . . . . . . . . . . . . . . Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.4.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.4.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . The Main Types of PCR . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 5 6 6 7 7 8 8 8 8

1.2 Reverse Transcription (RT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9

1.1.4

1.1.5

1.2.1 1.2.2

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Main Types of RT . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10 10

1.3 In Situ PCR/RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11

1.3.1

Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . In Situ Amplification Methods. . . . . . . . . . . . . . . . . . . . . . .

12 12 12 12

Direct In Situ PCR/RT-PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

1.4.1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.1.1 Amplification of a DNA Target Sequence (direct in situ PCR) . . . . . . . . . . . . . . . . . . . . . . . 1.4.1.2 Amplification of an RNA Target Sequence (direct in situ RT-PCR). . . . . . . . . . . . . . . . . . . . . Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.2.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.2.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

Indirect In Situ PCR/RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15

1.5.1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.1.1 Amplification of a DNA Target Sequence (indirect in situ PCR) . . . . . . . . . . . . . . . . . . . . . . 1.5.1.2 Amplification of an RNA Target Sequence (indirect in situ RT-PCR) . . . . . . . . . . . . . . . . . . . Advantages/Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.2.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.2.2 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

19

1.3.2 1.4

1.4.2

1.5

1.5.2

1.6

13 14 15 15 15

16 17 19 19 19

3

1.1

Polymerase Chain Reaction (PCR)

1.1 POLYMERASE CHAIN REACTION (PCR) PCR is used for synthesizing and amplifying, in vitro, specific nucleotide sequences of which only a very small number of copies are present in a given biological sample. It was described and named by Mullis et al. in 1987 (although the general principle had previously been set out by Khorana et al.). Since then, the technique has been considerably improved and has found many new applications. In 1990, in particular, Haase et al. developed an in situ PCR technique which combined the advantages and disadvantages of in situ hybridization and PCR, using cell suspension, to study the DNA of Lentivirus. This chapter, which is theoretical in orientation, deals first with the fundamental principles of liquid-phase PCR, then sets out the different in situ PCR techniques.

➫ Mullis, K. and Faloona, F., In: Methods in Enzymology, R. Wu, Ed., Academic Press, New York, 1987, Vol. 155, p. 335. ➫ Mullis was awarded the Nobel prize for chemistry in 1993. ➫ Kleppe, K. et al., J. Mol. Biol., 56, 341, 1971. ➫ Panet, A. and Khorana, H.G., J. Biol. Chem., 249, 5213, 1974. ➫ Haase, A. et al., Proc. Natl. Acad. Sci. U.S.A., 87, 4971, 1990.

1.1.1 Aim The aim of PCR is to amplify a specific sequence (the “target sequence”) of deoxyribonucleic acid to make numerous copies of it. This in vitro amplification, which is exponential, results in the synthesis of millions of copies of a specific segment of DNA.

➫ This sequence is determined by its two ends, 5′ and 3′ (see Figure 1.1).

1.1.2 The Target Sequence The target sequence is a known succession of nucleotides which is: • Present on one of the strands of a doublestranded DNA molecule, or • Part of the sequence of a single strand of DNA.

➫ This is the most common case (see Figure 1.1). ➫ If it comprises the entire sequence of the strand of DNA, it can be amplified by using a plasmid.

5

General Principles S = target sequence

S 5'

3'

Sc = sequence complementary to S 3'

5' Sc 3'

5'

S B

A 5'

3'

Sc D

A = 3′′ end of the target sequence B = 5′′ end of the target sequence C = 5′′ end of the sequence complementary to the target sequence D = 3′′ end of the sequence complementary to the target sequence Figure 1.1 The target sequence.

C

1.1.3 Overview After denaturation of the double-stranded DNA, PCR involves the hybridization and 3′ extension of a pair of primers: • An anti-sense primer • A sense primer At the end of this cycle, two identical copies of the DNA target sequence will have been obtained. It is the repetition of this cycle that allows a large number of copies to be obtained. This is what is known as a polymerization chain reaction (PCR). 1.1.3.1

➫ 2 copies, where n = the number of cycles. n

Primers

A primer hybridizes in a specific way to each of the two strands of DNA (S and Sc), thereby defining the sequence to be amplified. 5'

3' A

5'

3'

3'

5'

D

5'

➫ See Section 5.3.2. ➫ See Figure 1.1. ➫ The “anti-sense” primer is complementary and anti-sense to A. It corresponds to sequence C. ➫ The “sense” primer is complementary and anti-sense to D. It corresponds to sequence B. • • • Direction of the 3′ → 5′ polymerization. ▲

3'

6

➫ In the case of single-stranded DNA, only the hybridization of the anti-sense primer can take place during the first cycle. The sense primer hybridizes on the neosynthesized strand during the second cycle.

Figure 1.2 Positions of the primers at the ends of the target sequence.

1.1 1.1.3.2

Cycle

The PCR reaction comprises a three-stage cycle: • Denaturation • Hybridization • Extension

These three stages together make up an individual cycle. The repetition of this cycle is the basis of the PCR. It involves multiplying the number of copies of the target sequence. 1.1.3.3

Polymerase Chain Reaction (PCR)

➫ These are successive, consecutive stages. ➫ At 95°C, the double helix of the DNA matrix linearizes, and the two strands separate out. ➫ The primers then hybridize to the ends of the two strands of the DNA target sequence. ➫ DNA polymerase, attached to the 3′ ends of the primers, synthesizes the strands that are complementary to the two strands of DNA, in the presence of nucleotides, in a given reaction environment and precise temperature conditions. ➫ It is only at the end of the second cycle that the sequence of interest (a copy of the target sequence) is obtained (see Section 5.1). ➫ Each cycle doubles the number of copies of the target sequence and its complementary sequence.

Experimental conditions

Like other techniques in molecular biology, the success of PCR techniques depends on a number of rules being followed. ❶ Working environment. The experimentation must be carried out in sterile, or noncontaminating, conditions. This necessitates: • A room or bench reserved exclusively for PCR, if possible away from where the extractions take place • The cleanliness of the equipment, pipettes, centrifuge, and thermocyclers • The utilization of single-use consumables • The utilization of filter cones that prevent the propagation of contaminants through the air • Handling done in sterile conditions ❷ Verification of the quantity and quality of the DNA matrix. For this purpose, two types of densitometric measurement are carried out: • At 260 nm, to calculate how much of the nucleic acid to be amplified is present in the sample • At 280 nm, to calculate the quantity of contaminating proteins in the extract

➫ Given its high yield, a PCR requires particular vigilance with regard to the possible presence of contaminating DNA, which in fact is responsible for the majority of false-positive results observed in PCR.

➫

Gloves must be worn.

➫ One unit of DO260 nm corresponds to 50 µg of double-stranded DNA. ➫ The optimal DO260 nm/DO280 nm ratio is between 1.8 and 2. It indicates that the quality of the DNA matrix makes it suitable for PCR amplification.

❸ Division of the reagents. The reagents must be divided into aliquots as soon as possible. 7

General Principles ❹ Verification of the specificity of the two primers. ➎ Verification of the compatibility of the hybridization temperatures of the two primers. ➏ Verification thermocycler.

of

the

reliability

of

the

❼ The quality of the enzyme. The availability of DNA polymerase that can withstand very high temperatures means that the limitations of the PCR due to its lack of stability no longer apply. This has led to the automation of the technique, and its growing popularity.

➫ Each primer must be complementary to just one sequence in the genome, as determined by comparison with data banks (see Section 5.3.2). ➫ If the hybridization is to be specific, it must be carried out at a temperature determined by the Tm of the primers. These Tms must be very close together (see Section 4.3.1.3). ➫ Stability and reproducibility of the temperatures at which the different stages are carried out (see Section 4.4.1). ➫ See Section 5.3.3. ➫ Kogan, S.C. et al., N. Engl. J. Med., 317, 985, 1987. ➫ It should be possible to change the temperature rapidly, reliably, and without manual intervention.

1.1.4 Advantages/Disadvantages 1.1.4.1

Advantages

• The amplification of specific sequences • Rapidity • Amplification proportional to the number of cycles • Necessity to know only the sequences of the ends of the DNA sequence to be amplified • Differential analysis • Quantification possible 1.1.4.2

➫ The sequences are defined by their 3′ and 5′ ends. ➫ The cycle takes just a matter of minutes. ➫ It is limited, however, by the efficiency of the enzyme and the quantity of reagents. ➫ These sequences have to be known in order for the primers to be made. ➫ Amplification of particular sequence (see Section 1.1.5). ➫ Using a standard (see Section 1.1.5).

Disadvantages

• The tissue and cell structure are destroyed. • It is not possible to amplify RNA. • The amount of amplification is limited. • Absolute quantification is difficult.

➫ The technique is carried out after the extraction of the DNA. ➫ Reverse transcripton is necessary (see Section 1.2 and Chapter 4). ➫ This is always less than the theoretical yield, and varies from one experiment to another. ➫ This is due to the yield and the standard.

1.1.5 The Main Types of PCR There are several types of PCR: • Symmetrical PCR

8

➫ The sequence of interest and its complementary sequence are amplified simultaneously. ➫ The amplification is exponential. If the number of cycles is n, the number of copies n will be 2 .

1.2 • Asymmetrical PCR

• Nested PCR

• Semiquantitative PCR

• Quantitative PCR

Reverse Transcription (RT)

➫ Only the sequence of interest is amplified, but the amplified products are variable in size since the extension can stop either before or after the 5′ end of the target sequence. ➫ The amplification follows an arithmetic progression. If the number of cycles is n, the number of copies will be 2n. ➫ This is a double PCR in which a pair of primers situated on the amplified fragment during the first PCR makes possible a further amplification of a sequence, which is common but smaller than the amplified fragment, thus increasing the sensitivity of the PCR. ➫ This is used to estimate the number of copies of a sequence of interest that have been obtained, by comparison with the simultaneous amplification of a known synthesized sequence (i.e., the “mimic”), using the same reaction mixture. ➫ This is used to determine the number of copies of interest, by comparison with the simultaneous amplification of a range of dilutions of the mimic.

1.2 REVERSE TRANSCRIPTION (RT) PCR can only be used for DNA amplification. For an RNA target sequence to be amplified, it must first be turned into a complementary deoxyribonucleic acid (cDNA). A reverse transcription (RT) step is carried out, in the presence of: • A primer, which may be of different types • Triphosphate nucleotides • A specific enzyme, i.e., reverse transcriptase

➫ See Chapter 4. ➫ See Section 4.3.1. ➫ See Section 4.3.3.

The result is a DNA copy (i.e., a transcription of RNA into cDNA). This neosynthesized cDNA can then be amplified by PCR. ❑ Advantages Reverse transcription makes it possible to: • Amplify a given type of RNA • Amplify different types of RNA

• Stabilize a solution of different types of RNA by turning them into cDNA

➫ RNA cannot be amplified directly by PCR. ➫ Depending on the type of primer: all the different types of RNA, or just a certain number, can be transcribed into cDNA (see Section 4.3.1). ➫ An RNA solution is always highly unstable, and is best conserved in the form of cDNA. 9

General Principles ❑ Disadvantages • This step is difficult to evaluate. • The yield depends on a large number of factors.

➫ See Section 9.3. ➫ These factors are related to the primers, the reliability of the enzyme, etc. (see Chapter 4).

1.2.1 Overview 5'

3'

5'

AAAAA

➫ RNA

AAAAA

➫ The hybridization of the primer

3'

+ RT

5'

3' AAAAA

RT

3'

5'

➫ The extension of the primer in the presence of the enzyme (reverse transcriptase) and the deoxynucleotides (dATP, dCTP, dGTP, and dTTP) ➫ cDNA Figure 1.3 Reverse transcription (RT).

1.2.2 The Main Types of RT There are three different types of RT, corresponding to the three types of primer: • Poly (T) primers • Random primers • Specific primers These different types of RT can be combined with different PCRs to obtain a complete range of methods for the amplification of RNA. • • • •

10

Symmetrical and asymmetrical PCR Nested PCR Semiquantitative and quantitative PCR Differential display PCR (ddPCR)

➫ All the different types of poly (A) RNA retrotranscribed into cDNA (see Section 4.3.1.1) ➫ Nonspecific transcriptions of many types of RNA into cDNA (see Section 4.3.1.2) ➫ The specific transcription of the target sequence (see Section 4.3.1.3) ➫ See Section 1.5.

➫ Only the RNA that remains after differential hybridization will be retrotranscribed, then amplified.

1.3

In Situ PCR/RT-PCR

1.3 IN SITU PCR/RT-PCR PCR is used in situ to visualize, in cell compartments, DNA or mRNA that is present only in a small number of copies. The results depend on the compromise that is established between ➊ Preserving • The sequence of interest

• The tissue structure

• The cell structure

❷ And facilitating the accessibility of • The sequence of interest

• The amplified products

❸ While avoiding • The diffusion of the amplified product

➫ The success of this technique depends on the PCR rules, along with all the constraints linked to the preservation of morphology.

➫ This is present only in a small number of copies, which must be preserved in suitable conditions, i.e., without breaks or digestion by RNase (see Section 2.3). ➫ It is essential to link the detected expression to the typical organization of the organ. Fixation is an important step (see Chapter 2). ➫ This is necessary to identify the cell type responsible for the detected expression, and to limit the diffusion of the amplified product; the cell constitutes a veritable PCR chamber. ➫ PCR/RT-PCR is preceded by a set of pretreatments of the tissue or cells (see Chapter 3). These are indispensable to the penetration of the reagents. ➫ In most cases, the detection of the amplified products requires an immunocytological reaction that uses immunoglobulins of high molecular weight (see Chapter 7). This step is generally not a problem, since the tissue sections or cells are subjected to a certain number of chemical treatments (proteinase, DNase, etc.) and physical treatments (high temperature), which ensure the permeability of the membranes. ➫ Excessive membrane permeability leads to the diffusion of the amplified products, which is the main cause of false positives.

➫ The tissue or cells are fixed (see Chapter 2). Figure 1.4 Schematic representation of a fixed cell.

11

General Principles ➫ Permeabilized cytoplasmic and nuclear membranes.

➫ Accessible DNA or RNA target sequences Figure 1.5 A pretreated cell.

1.3.1 Advantages/Disadvantages 1.3.1.1

Advantages

• This method can be used to amplify a given sequence of nucleic acid, either DNA or mRNA. • It can be used to visualize the amplified product in situ. • It can be used to identify the tissue structure. • It can be adapted to the ultrastructural scale. 1.3.1.2

➫ PCR or RT-PCR ➫ By immunocytological detection ➫ Characterization of the different positive and/or negative tissue components ➫ See Chapter 8.

Disadvantages

• Limited efficiency of the amplification

• Diffusion of the amplified products

• The partial destruction of morphology

• The necessity for specific equipment

➫ The constraints imposed by the tissue environment (proteins, lipids, sugars) interfere with the PCR reagents. ➫ This theoretical question can only be resolved by the presence of internal controls (i.e., positive and negative cells on the same tissue section) and the presence of a positive control. ➫ This is due to the necessary pretreatments and to variations in temperature during the amplification cycles. ➫ A thermocycler that will take slides.

1.3.2 In Situ Amplification Methods There are two main types: • Direct reaction • Indirect reaction

➫ See Sections 5.3.1.3, 5.3.2.7, and 5.5.1.1. ➫ See Chapter 5.

which are applied to • Tissue, or • Cells in culture both in light microscopy and electron microscopy. 12

➫ See Chapter 5. ➫ See Section 5.5.6. ➫ See Chapter 5. ➫ See Chapter 8.

1.4

Direct In Situ PCR/RT-PCR

1.4 DIRECT IN SITU PCR/RT-PCR The aim of this approach is to obtain an amplified product by the incorporation of a label in the course of the amplification phase. This allows the amplified products to be detected by means of an immunocytological reaction.

➫ The label is either coupled to the deoxynucleotide triphosphates that are incorporated during the amplification process or present on the primers that define this amplification. ➫ These labels are generally antigens or, in rare cases, radioactive isotopes (see Section 5.3).

1.4.1 Overview 1.4.1.1

Amplification of a DNA target sequence (direct in situ PCR) ① Denaturation of the nucleic acids

② Amplification of the DNA target sequence in the presence of: — Labeled primers — , or — Nucleotides carrying the label 

③ The DNA target sequences amplified and directly labeled

Figure 1.6 Direct in situ PCR. Either the amplified product is directly visible, through the emission of a radioactive label (Figure 1.7), or the incorporated antigenic label is detected immunocytologically (Figure 1.8).

➫ See Chapter 7.

13

General Principles ➫ The macroautoradiographic film or nuclear emulsion is apposed to the tissue sections or cells (see Section 7.3). ➫ The emitted radiation is recorded on a photographic support. ➫ The signal corresponds to the amplified DNA. Figure 1.7 Autoradiographic detection. ➫ The immunoglobulin attaches to the label, forming an antigen/antibody complex that is either directly visible (fluorescent antibody) or detected by an enzymatic reaction (alkaline phosphatase or peroxidase) (see Section 7.2). Figure 1.8 Immunocytological detection of an incorporated antigen. 1.4.1.2

Amplification of an RNA target sequence (direct in situ RT-PCR) ➫ The poly (T) random or specific anti-sense primer hybridizes to the strand of RNA (see Chapter 4). ➫ The reverse transcriptase synthesizes cDNA (see Section 4.3.3).

Figure 1.9 Reverse transcription of RNA. ① Amplification of a cDNA sequence in the presence of: — Labeled primers — , or — Nucleotides carrying the label  ➫ See Chapter 5.

② Direct marking of the amplified product

Figure 1.10 Amplification of retrotranscribed cDNA. 14

1.5 As with a direct PCR, the amplified product obtained by a direct RT-PCR is detected by autoradiography or an immunocytological reaction.

Indirect In Situ PCR/RT-PCR

➫ Radioactive label (see Section 7.3). ➫ Antigenic label (see Section 7.2). ➫ The macroautoradiographic film or nuclear emulsion is apposed to the tissue section or cells (see Section 7.3). ➫ The emitted radiation is recorded on a photographic support. ➫ The signal corresponds to the amplified RNA. Figure 1.11 Autoradiographic detection. ➫ Immunoglobulin attaches to the label to form an antigen/antibody complex that is directly visible (fluorescent antibody) or detectable by an enzymatic reaction (alkaline phosphatase or peroxidase) (see Section 7.2). Figure 1.12 Immunocytological detection of an incorporated antigen.

1.4.2 Advantages/Disadvantages 1.4.2.1

Advantages

• These methods are used to visualize amplified products directly. • They are rapid. • They are extremely sensitive. 1.4.2.2

➫ Whether DNA (direct in situ PCR) or RNA (direct in situ RT-PCR). ➫ Hybridization is not necessary. ➫ All the neosynthesized products are labeled.

Disadvantages

• It is difficult to be sure that the reaction has taken place correctly. • A large number of false positives may occur.

➫ See Section 9.3. ➫ See Section 9.4.

1.5 INDIRECT IN SITU PCR/RT-PCR The aim of this approach is the specific detection of neosynthesized amplified products by a further hybridization step. The probes carrying the label are either radioactive or antigenic, and form hybrids with the sequence of interest. These hybrids are visualized by autoradiography or detected by immunocytology.

➫ This hybridization uses two probes, each complementary to one of the strands of the amplified product (see Chapter 6). ➫ Unlike the direct amplification methods, the indirect methods mostly use a radioactive label, given that the risk of contamination is lower (see Chapter 11). ➫ See Chapter 7. 15

General Principles The specificity of the probes is essential to the characterization of the amplified products. A specific hybridization can compensate for a lower degree of specificity in the PCR.

➫ Their specificity must be total, and must not allow any nonspecific hybridization to take place in the optimal hybridization conditions. ➫ The length of the probes can be increased to the point where total specificity is achieved.

1.5.1 Overview 1.5.1.1

Amplification of a DNA target sequence (indirect in situ PCR) ① Denaturation of the nucleic acids

② Amplification carried out in the presence of unmodified nucleotides or unlabeled primers (see Chapter 5)

③ Amplified DNA sequences

④ Hybridization with a pair of labeled primers (see Chapter 5)

16

1.5

Indirect In Situ PCR/RT-PCR

⑤ Labeled hybrids

⑥ Immunocytological detection (antigenic label) ➫ See Section 7.2.

⑦ Autoradiographic detection (radioactive label) ➫ See Section 7.3.

Figure 1.13 Indirect in situ PCR. 1.5.1.2

Amplification of an RNA target sequence (indirect in situ RT-PCR) ① Reverse transcription of mRNA (see Chapter 4)

② Amplification of retrotranscribed cDNA in the presence of unlabeled markers or unmodified nucleotides (see Chapter 5)

17

General Principles ③ Sequences of the amplified, unlabeled cDNA of interest

④ Hybridization with a pair of labeled primers (see Chapter 5)

⑤ Labeled hybrids

⑥ Autoradiographic detection (radioactive label) ➫ See Section 7.3.

⑦ Immunocytological detection (antigenic label) ➫ See Section 7.2.

Figure 1.14 Indirect in situ RT-PCR.

18

1.6

Conclusion

1.5.2 Advantages/Disadvantages 1.5.2.1

Advantages

• The characterization of the amplified product. • With this type of method, the specificity of the reaction is much higher and easier to check. • The use of unlabeled primers and nucleotides. 1.5.2.2

➫ It is the specificity of the probes that ensures this. ➫ See Chapter 9. ➫ Low cost

Disadvantages

• Indirect methods take longer than direct methods. • They are also less sensitive.

➫ See Chapter 5.

1.6 CONCLUSION For a given sequence of nucleic acid, whether DNA or RNA, the choice between the different in situ amplification methods, as well as between visualization by an immunocytological reaction or by autoradiography, will depend on: • The nature of the target sequence • The concentration of this sequence • The possibility of nonspecific reactions The characteristics of the different approaches can be summarized as follows:

➫ The estimated number of copies per cell

Target

Technique

Method

Specificity

Sensitivity

DNA

PCR

Direct Indirect

+ ++

++ +

RNA

RT-PCR

Direct Indirect

+ ++

++ +

19

Chapter 2 Preparation of Samples

Contents

CONTENTS 2.1 Tissue Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25

2.1.1 Sampling Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25

2.1.2 Diagram of the Different Steps. . . . . . . . . . . . . . . . . . . . .

26

2.1.3 Fixation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

26

2.1.3.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3.2 Fixation Parameters . . . . . . . . . . . . . . . . . . . . . . 2.1.3.3 Fixatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3.3.1 Cross-Linking Fixatives . . . . . . . . . . 2.1.3.3.2 Precipitating Fixatives . . . . . . . . . . . 2.1.3.3.3 Fixative Mixtures . . . . . . . . . . . . . . . 2.1.3.4 Fixation Protocols . . . . . . . . . . . . . . . . . . . . . . .

26 26 27 28 30 30 31

2.1.4 Frozen Fixed Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

31

2.1.4.1 Cryogenic Agents. . . . . . . . . . . . . . . . . . . . . . . . 2.1.4.2 Freezing Method . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4.2.1 Cryoprotection . . . . . . . . . . . . . . . . . 2.1.4.2.2 The Freezing Operation . . . . . . . . . . 2.1.4.2.3 Conservation . . . . . . . . . . . . . . . . . . 2.1.4.3 Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4.4 Advantages/Disadvantages . . . . . . . . . . . . . . . .

32 32 32 33 34 34 36

2.1.5 Fixed Paraffin-Embedded Tissue . . . . . . . . . . . . . . . . . . .

36

2.1.5.1 2.1.5.2 2.1.5.3 2.1.5.4

Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paraffin Embedding . . . . . . . . . . . . . . . . . . . . . . Making Sections . . . . . . . . . . . . . . . . . . . . . . . . Advantages/Disadvantages . . . . . . . . . . . . . . . .

36 36 38 39

2.1.6 Nonfixed Frozen Tissue . . . . . . . . . . . . . . . . . . . . . . . . . .

39

2.1.6.1 2.1.6.2 2.1.6.3 2.1.6.4

Freezing Protocols . . . . . . . . . . . . . . . . . . . . . . . Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . . Fixation of Frozen Sections . . . . . . . . . . . . . . . . Advantages/Disadvantages . . . . . . . . . . . . . . . .

39 39 39 40

2.2 Cultures/Cellular Smears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40

2.2.1

Cell Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40

2.2.1.1 The Different Possibilities . . . . . . . . . . . . . . . . . 2.2.1.2 Diagram of the Different Steps . . . . . . . . . . . . .

40 41

23

Preparation of Samples 2.2.2

Cell Cultures on Slides . . . . . . . . . . . . . . . . . . . . . . . . . . .

42

2.2.2.1 Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.2.1 Cross-Linking Fixatives . . . . . . . . . . 2.2.2.2.2 Precipitating Fixatives . . . . . . . . . . . 2.2.2.2.3 Fixation Protocols . . . . . . . . . . . . . . 2.2.2.3 Advantages/Disadvantages . . . . . . . . . . . . . . . .

42 43 43 43 43 44

2.2.3 Cellular Smears. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

44

2.2.3.1 Obtaining Samples . . . . . . . . . . . . . . . . . . . . . . 2.2.3.2 Fixation Protocol . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3.3 Advantages/Disadvantages . . . . . . . . . . . . . . . .

44 44 45

Cellular Pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45

2.2.4.1 Obtaining Samples . . . . . . . . . . . . . . . . . . . . . . 2.2.4.2 Fixation Protocol . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4.3 Advantages/Disadvantages . . . . . . . . . . . . . . . .

45 45 45

2.2.4

24

2.1

Tissue Samples

On what types of biological material can in situ PCR and RT-PCR be performed? What precautions have to be taken in collecting such material, and what are the fixation conditions that must be adhered to? This chapter provides answers to such questions. Whatever the origin of the biological material, the objective of the methods presented here is to ensure that nucleic acids are preserved, along with tissue and cell morphology.

2.1 TISSUE SAMPLES In situ PCR can be carried out on various types of sample, from complete organs to biopsy material, of either human or animal origin.

➫ In situ PCR is rarely carried out on vegetable tissue, which requires a specific type of preparation.

2.1.1 Sampling Conditions The sampling conditions are strict, and must prevent all possibility of contamination by extraneous RNase or DNase. The sampling procedure must be rapid, and the subsequent operations carried out as soon as possible. These are:

• Fixation, followed by: — Freezing ®

— Paraffin or Paraplast embedding

• Freezing, without fixation, followed by: — Fixation on slides

➫

Gloves must be worn.

➫ Sterilized or single-use equipment (flasks, tubes, surgical instruments). ➫ This requirement is often not respected in the case of surgical resections carried out for the purpose of anatomopathological diagnosis. The expression of the results must take account of this fact. ➫ On the use of a cryostat to obtain sections, see Section 2.1.4. ➫ To obtain paraffin-embedded tissue sections, see Section 2.1.5. Paraffin has now been largely replaced by the synthetic embedding medium ® Paraplast . ➫ To obtain sections on a cryostat

25

Preparation of Samples

2.1.2 Diagram of the Different Steps

Tissue

Freezing

Fixation

Freezing

Paraffin embedding

Frozen sections

Frozen sections

Sections

Fixation

2.1.3 Fixation 2.1.3.1

Overview

Fixation is essential to the success of in situ PCR. Its purposes are: • To shut down the cellular machinery, but in a state that is as close as possible to that of the living organism • To preserve the organization of the tissue, along with the shape and volume of the cell • To prevent autolysis or bacterial attacks • To inactivate lysosomial enzymes and endogenous RNase

➫ The fixation procedure must allow the in situ amplification of the DNA or RNA to be carried out in the place where it was synthesized.

It must be carried out quickly: • In vivo • Immediately after the obtaining of the fresh sample 2.1.3.2

Fixation parameters

The satisfactory fixation of tissue depends on a number of factors:

26

➫ Fixation by perfusion ➫ Fixation by immersion

2.1 ❶ The diffusion of a fixative through tissue can be expressed by the formula: d = K t which shows that the penetration of the fixative, d, is proportional to the square root of the fixation time, t, with K the coefficient of diffusion of the particular tissue type, representing the depth, in millimeters, to which the fixative penetrates in 1 hour. ❷ Temperature It is generally advisable to carry out the fixation procedure at room temperature.

❸ Fixative concentration Each fixative has its own maximal efficiency concentration. Higher or lower concentrations give rise to artefacts that are difficult to interpret. ❹ pH Fixatives are used in buffered solutions at a pH of between 7.2 and 7.4. The pH has an effect on the molecular conformation of various cell components, and too low or too high a pH can affect reactions between proteins and aldehydes. ❺ The osmolarity of the mixture (buffer + fixative) must correspond to normal physiological osmotic pressure. ❻ Fixation time varies between 2 and 24 hours, depending on the size and origin of the biological samples.

2.1.3.3

Tissue Samples

➫ The coefficient of diffusion, K, depends on the tissue in question. It is generally measured on a gel, or on homogeneous tissue such as that of the liver. The values are generally lower for tissue than for gels, since the different constituents of tissue act as barriers to the penetration of the fixative.

➫ While lower temperatures would reduce the effects of autolysis related to anoxia, they would also severely hinder the penetration of the fixative, particularly in the case of aldehydes. ➫ If the concentration is too low, morphology will not be properly preserved. ➫ Too high a concentration leads to overfixation, which reduces the accessibility of the nucleic acids due to an increase in protein– DNA bonds. ➫ Phosphate or cacodylate buffers are the most common. ➫ It is important to check that the fixative and the buffer do not interact with each other. ➫ The osmotic pressure can be adjusted by the addition of sodium chloride, or compounds such as sucrose. ➫ It should not exceed 24 hours. Beyond this there are problems due to overfixation, and it becomes difficult to entirely eliminate the fixative, even with prolonged washing.

Fixatives

Fixatives can be divided into three groups, according to their mode of action on proteins: • Cross-linking fixatives

• Precipitating fixatives • Fixative mixtures

➫ Aldehydes, e.g., formaldehyde and glutaraldehyde, which form intra- and intermolecular bridges by reacting with the amines in the lateral chains of the peptides. ➫ Alcohol and acetone, which are “coagulants,” cause proteins to precipitate. ➫ A number of different fixatives can be combined in such a way that their advantages are cumulative and their disadvantages are canceled out. 27

Preparation of Samples ➫ Most of these mixtures contain mercury salts, and are suitable for trichromic stainings, but should not be used for in situ PCR. 2.1.3.3.1

CROSS-LINKING FIXATIVES

❶ Formaldehyde or formic aldehyde is a gas which, in aqueous solution, produces methylene glycol. OH

H C

O

H

HCHO + 2H2O

H

C

OH

➫ This is “commercial formalin,” which is supplied at a concentration of 37%. Note: The solution may contain stabilizing agents such as methanol (10 to 15%) or calcium. ➫ It is toxic if inhaled, or if it comes into contact with the skin or the mucous membranes. It should be handled under a ventilated hood.

H

OHCH2OH

• Mode of action: The first stage in the reaction of this monoaldehyde with proteins involves the formation of amino-methylol groups, starting with their free amine groups, which then condense into methylene (–CH2–) bridges. • Action on nucleic acids: Aldehyde groups react with nucleic acid template to form DNA–DNA, RNA–RNA, RNA/DNA–histone protein cross-links. • Preparation: — Neutral buffered formalin — Saline formalin

Figure 2.1 Formula of the formaldehyde. ➫ The maximum number of these bridges occurs at a pH of between 7.5 and 8.

➫ Most of these bridges are eliminated by postfixative treatment of the tissue (washing, dehydration) and a suitable proteolytic digestion process. ➫ See Appendix B4.1. ➫ See Appendix B4.1.

❑ Advantages • Rapid penetration

• A high degree of conservation of proteins, which satisfies morphological requirements

• The molecular network resulting from the methylene cross-links, which protects RNA from being broken down by RNase ❑ Disadvantages • Prolonged fixation by aldehydes causes breaks to occur in the DNA chain, and these may become extension sites through TaqDNA polymerase. 28

➫ It is, however, advisable to use small fragments of tissue to obtain good-quality fixation (the penetration speed is of the order of 1 mm/h). ➫ A satisfactory degree of conservation of cellular structures is indispensable if the diffusion of amplification products outside the cell, i.e., in the amplification medium, is to be avoided.

2.1 • These conformational changes reduce the efficiency of hybridization with homologous DNA. ❷ Paraformaldehyde This is a polymer of formaldehyde. It is prepared in a buffered solution without a stabilizing additive.

➫ This is the most widely used fixative for in situ PCR and RT-PCR techniques. ➫ This is its main advantage compared to formaldehyde. Otherwise, it has the same properties, advantages, and disadvantages as formaldehyde.

OH

H C

Tissue Samples

H

O n

H

C

n

OH

H

Figure 2.2 Formula of paraformaldehyde.

• Preparation: — Paraformaldehyde 40% — Paraformaldehyde 4%

➫ Stock solution (see Appendix B4.3) ➫ Working solution (see Appendix B4.3)

❑ Advantages • Identical to those of formaldehyde • A pure solution, without preservatives

➫ See above. ➫ The absence of precipitating fixatives

❑ Disadvantage • A limited conservation time ❸ Glutaraldehyde O

O C

CH2

H

CH2

CH2

➫ 1 month at 4°C ➫ This is the best fixative in terms of morphological conservation, but it should not be used for in situ PCR or RT-PCR techniques at concentrations higher than 0.05%.

C H

• Mode of action: It forms long-chain polymers, providing a high level of conservation of tissue structures. • Preparation: A weak (0.025–0.05%) solution of glutaraldehyde can be added to a 3.5% paraformaldehyde solution.

Figure 2.3 Formula of glutaraldehyde.

➫ Working solution (see Appendix B4.3)

❑ Advantage • It works rapidly: Glutaraldehyde at 1% fixes proteins and peptides in 10 s, and free amino acids in 1 min.

➫ The lowest concentration that has a fixative effect is 0.025%.

❑ Disadvantage • The cross-links, which make nucleic acids less accessible, are not reducible.

➫ Cross-links considerably reduce the penetrability of tissue, even after intense proteasic digestion.

29

Preparation of Samples 2.1.3.3.2

PRECIPITATING FIXATIVES

❶ Ethyl alcohol (CH3CH2OH)

➫ Alcohols conserve nucleic acids very well, but should not be used for the fixation of tissue intended for in situ PCR due to the poor conservation of structures at all concentrations. ➫ Generally used only for fixing frozen sections and cellular smears. It should definitely not be used for fixing tissue.

❷ Methyl alcohol (CH3OH) ❸ Acetone

2.1.3.3.3

FIXATIVE MIXTURES

❶ Bouin’s fixative: A mixture of formalin, picric acid and acetic acid. It conserves tissue and cell morphology very well, but causes nucleic acids to break down and change their conformation, thus making them less accessible.

❷ Carnoy’s fixative: A mixture of ethyl alcohol, chloroform, and acetic acid. ❸ Zenker’s fixative: A mixture of acetic acid, mercury bichloride, potassium bichromate, and disodium sulfate.

➫ This fixative should therefore, in principle, not be used in hybridization or amplification techniques. ➫ Although this fixative creates problems for those who want to make retrospective use of diagnosed clinical cases, it is the one that is most widely used by anatomopathologists in France. Some authors have, however, reported that undesirable effects are kept to a minimum if the fixation time does not exceed 2 hours. ➫ It is strongly advised that this fixative not be used for in situ PCR, since acetic acid hydrolyzes nucleic acids. ➫ Mercury salts can form complexes with nucleic acids, thus limiting the accessibility of the latter.

Properties of Fixatives Penetration

Conservation of Nucleic Acids

Morphological Preservation

Methanol

+++

+++

+

Ethanol

+++

+++

+

Acetone

+++

++

+

Formaldehyde

++

++

++

Paraformaldehyde

++

++

++

+

+

+++

Paraformaldehyde and glutaraldehyde

++

++

+++

Other mixtures

++

To be tested

Fixative

Glutaraldehyde

*

By in situ hybridization with a poly (T) probe.

30

*

+++

2.1 2.1.3.4

Tissue Samples

Fixation protocols

❶ Fixation by immersion • The samples should ideally not exceed 5 mm in diameter, and should immediately be immersed in a suitable fixative.

• Fixation time

4–24 h

rt

• Rinses in a buffer solution (phosphate or cacodylate)

2 × 15 min

❷ Fixation by perfusion, followed by fixation by immersion. After the animal has been anesthetized, and the perfusion system put in place:

• Rinse rapidly with a buffer solution. • Inject the fixative (generally paraformaldehyde in a phosphate buffer).

40 ml/min

➫ This is the most commonly used fixation method. ➫ The samples can be stored in a cassette bearing an identifying label until the embedding process is carried out. ➫ Paraformaldehyde and buffered formalin are the most commonly used fixatives. See Section 3.1.3.1.3. ➫ This depends on the size of the samples. ➫ It is advisable to cut large samples again after 1–2 hours of fixation to facilitate the penetration of the fixative. ➫ rt = room temperature. Low temperatures considerably reduce the speed of penetration of aldehydes in particular. ➫ See Appendix B3. ➫ Theoretically, rinsing time should be proportional to fixation time, but in the case of in situ PCR and RT-PCR prolonged rinsing can reduce tissue integrity, and should be avoided. ➫ This is recommended for the study of the central nervous system, and for delicate tissue. ➫ The heart is exposed by opening the rib cage. A canula is introduced into the aorta after the opening of the left ventricle. It is connected by a catheter to a peristaltic pump. An incision is made in the right ventricle so that the fluid can circulate by perfusion. ➫ For a rat, around 50 ml of buffer is perfused at body temperature. ➫ Around 500 ml of fixative in solution are necessary to perfuse a rat. ➫ The flow rate must be kept steady if serious tissue lesions are to be avoided.

• Remove the organs to be studied rapidly. • Immerse these organs, 1–2 h or fragments of organs, 4°°C, or in the same fixative. rt

2.1.4 Frozen Fixed Tissue After fixation, biological samples can be frozen for conservation and storage purposes. Homogeneous freezing preserves nucleic acids in situ and ensures their structural conservation; these are the two factors that essentially determine the results of in situ PCR and RT-PCR.

➫ The hardening of the samples facilitates the subsequent cutting of sections. ➫ Visualization of amplified products in the conserved cellular compartment. 31

Preparation of Samples 2.1.4.1

Cryogenic agents

❶ Liquid nitrogen (−196°C)

➫ Widely used as a cryogenic agent, given that it is easy to obtain and store.

❑ Advantages • Liquid nitrogen can be used for storing samples after freezing. • It is not particularly dangerous.

➫ Use adequate containers, i.e., not airtight (risk of explosion). ➫ Storage containers must be kept in wellventilated surroundings so that the evaporating nitrogen gas does not reduce the relative percentage of oxygen in the surrounding air.

❑ Disadvantage • Insulation effects

❷ Isopentane (methyl-2-butane) cooled in liquid nitrogen (−160°C)

➫ The boiling temperature of liquid nitrogen is very close to that of its liquefaction, so that the immersion of a warm object causes it to boil. The resulting nitrogen gas forms an insulating layer around the sample (chelefaction phenomenon), thereby reducing the cryogenic properties of the liquid nitrogen. ➫ This avoids the insulation problem.

❑ Advantage • Its temperature remains constant during the freezing procedure.

❸ Dry ice (−78°C)

2.1.4.2 2.1.4.2.1

Freezing method CRYOPROTECTION

Prior to freezing samples that have been fixed and rinsed in a buffered solution, measures should be taken to limit damage caused by the formation of ice from water within cells. Water-soluble cryoprotectant can be used for this purpose.

❶ Saccharose or α -D-Glucopyranosyl β -D-fructofuranosode • Formula C12H22O11 32

➫ Below −160°C, isopentane becomes viscous. A thin layer of viscous isopentane in the bottom of a receptacle dipped in liquid nitrogen will confirm that the latter is at the right temperature. ➫ This is a contact cryogen. It does not produce homogeneous freezing, and should be avoided. ➫ It can, however, be used for flat samples.

➫ For a cryoprotectant to be effective, it must diffuse rapidly, which means that it must be of low molecular weight. It also needs to be a non-electrolyte, so that it can penetrate the cell easily and mix with saline solutions. ➫ Chemically neutral

2.1 • Mw • Concentration

342.3 30% in 0.1 M phosphate buffer, pH 7.4

❷ Glycerol • Formula • Mw • Concentration

C3H8O3 92.09 5–10%

❸ Dimethylsulfoxide (DMSO) • Formula • Mw • Concentration

C2H6SO 78.13 5–15%

2.1.4.2.2

Tissue Samples

➫ The concentration can be adjusted according to the tissue. If the latter contains a lot of water, successive baths of increasing concentration are advisable. ➫ Also nontoxic for most cells. In spite of its low membrane permeability, it is a very good cryoprotectant. ➫ It is perfectly miscible with water above its fusion temperature (+18°C). ➫ Toxic. ➫ A good cryoprotectant. As an organic solvent it can, however, have an effect on cell membranes.

THE FREEZING OPERATION

❶ After coating with O.C.T. mounting medium ® (Tissue-Tek ) • Place the sample in a rubber mold. • Cover with O.C.T.

➫ Highly recommended for samples of small size, which risk thawing out while being attached to the slide holder. ➫ This facilitates the cutting of the sections. ① Coating medium ② Sample ③ Embedding mold

1 2 3

• Dip in liquid nitrogen.

∼1 min −196°°C

Figure 2.4 Embedding O.C.T. mounting medium. ➫ Or in isopentane cooled in liquid nitrogen. ① Sample ② Isopentane ③ Liquid nitrogen

Figure 2.5 Freezing in isopentane cooled in liquid nitrogen.

33

Preparation of Samples • Remove the samples from the molds and quickly place them in cryotubes. ❷ Without coating: There are several possible techniques. ∼1 min • Immersion of the samples in −160°°C isopentane cooled in liquid nitrogen • Direct immersion in liquid nitrogen

∼1 min −196°°C

• Freezing in vapor from liquid nitrogen after total immersion, to simulate progressive freezing • Freezing by contact with dry ice

2.1.4.2.3

➫ This is the most commonly used technique. At −160°C, the risk of breaks is small, and the thermal exchanges lead to homogeneous freezing. ➫ Up to the disappearance of the insulation phenomenon ➫ The risk of breaks is high, especially for large samples. ➫ Samples can be placed in tubes, or wrapped in aluminum foil. In such conditions, they should be allowed to float on the surface of the liquid nitrogen. ➫ This procedure cuts down the number of breaks. ➫ This technique should be considered a “second best” option.

CONSERVATION −196°°C −80°°C

➫ No time limitations ➫ At −80°C the preservation will always be of shorter duration.

❶ Mounting the sample on the cryostat support

➫ RNase-free conditions must be observed.

• In liquid nitrogen • In the freezer

2.1.4.3

Frozen sections

• Place the support on the arm of the cryostat (−20°C), and cover it with a layer of O.C.T. • Rapidly position the sample according to the desired plane of the cut before covering it completely with the coating medium.

• Wait for the temperature of the whole system (sample, support, and knife) to reach equilibrium (≈ −20°C).

➫ The sample must never thaw out during this step. ➫ If the sample was embedded in O.C.T. before freezing, it is sufficient to adhere the block to the chilled support with a thin layer of the mounting medium. ➫ This takes around 15 min with the cryostat closed. ① Cryostat support ② Frozen sample

Figure 2.6 Mounting the sample on the cryostat support. 34

2.1

Tissue Samples

❷ Making frozen sections • The sections must be of regular thickness, between 7 and 10 µm.

• The sections are placed on slides that have been treated with 3-amino-propyl-triethoxysilane and sterilized in an oven for 3 hours at 180°C.

➫ The quality of the sections will have an effect on the final results. ➫ The cryostat temperature should be adapted to the hardness of the sample. ➫ One, two, or three sections are placed on each slide. ➫ See Appendix A3. ➫ With the Perkin-Elmer thermocycler, specially made slides must be used. Cover disks and cover clips are used for amplification.

• The proper spreading and adhesion of the section result from the difference in temperature between the section and the slide. ① Frozen sample on support ② Knife

1 3

③ Section

④ Transfer of the section on the slide

Figure 2.7 Production of frozen tissue sections. ❸ Drying • At room temperature

• In a vacuum jar ❹ Storage In hermetically sealed boxes, with a desiccant (silicagel)

1–4 h

➫ Complete dehydration is necessary for the adhesion of the sections to the slides and for conservation of the nucleic acids (RNase and DNase become active only in the presence of water).

1h −20°°C or −80°°C

➫ The slides remain usable for several months, or even years. ➫ It is important to leave the box at room temperature for around 2 h before opening it, to avoid the risk of condensation, and thus the rehydration of the sections. 35

Preparation of Samples ➲ Following step • Proteolytic pretreatments 2.1.4.4

➫ See Section 3.5.

Advantages/disadvantages

❑ Advantages ➫ Optimal preservation of nucleic acids. ➫ Fixation by aldehydes allows tissue and cellular structure to be conserved, which is indispensable to obtaining satisfactory results.

• Sensitivity • Morphology

❑ Disadvantages • Storage conditions are strict, both for the samples and the slides. • Frozen tissue is delicate, even when fixed, and is less resistant than paraffin-embedded tissue to drastic treatments such as proteolysis, or the high temperatures involved in PCR.

➫ Risk of thawing

2.1.5 Fixed Paraffin-Embedded Tissue Paraffin embedding hardens samples, which makes it easier to obtain histological sections. 2.1.5.1

Fixation

The best results are obtained with aldehydic fixatives. • Neutral buffered formalin 2–24 h

• Paraformaldehyde 4%

2–24 h

The amplification of infinitesimal quantities of RNA or DNA allows the use of samples whose nucleic acids have been largely destroyed by the Bouin’s fixative.

2.1.5.2

➫ See Section 3.1.3.3.1. ➫ Time depends on the size of the sample. ➫ The fixation of animal samples by perfusion is not indispensable (see Appendix B4.1.1). ➫ Time depends on the size of the sample (see Appendix B4.3). ➫ It is generally considered that after 8 h of fixation in Bouin’s fixative the signal is very weak, and that after 15 h it has disappeared. ➫ Their presence can be checked by hybridization with a poly (T) probe.

Paraffin embedding

Figure 2.8 Paraffin embedding cassette. 36

2.1 ❶ Washing samples in a buffer solution

2–3 × 15 min

❷ Dehydration in alcohol baths 1–2 h each of increasing strength: 70°, 95°, 100° ❸ Impregnation 1–2 h • A bath of xylene or methyl cyclohexane (a solvent for paraffin) • A bath of solvent/paraffin 1–2 h mixture (v/v) 56°°C • Two paraffin baths 1–4 h each 56°°C ❹ Embedding • Place the sample, according to the desired orientation, in a mold covered with a thin layer of liquid paraffin. — The mold fits onto the lower part of the box in which the sample has been kept after fixation; or

Tissue Samples

➫ All trace of fixative must be eliminated. Most of the bridges created by aldehydes can be reduced by washing in an aqueous medium.

➫ Paraffin liquefaction temperature

➫ After the combination of block and cover has been removed from the mold, it is attached directly to the microtome arm.

Figure 2.9 Positioning the sample in the paraffin embedding mold. — The use of Leukart bars on a flat surface (e.g., an earthenware tile or a metal plate) • Cover with liquid paraffin.

➫ In this way, large samples (e.g., entire organs or embryos) can be embedded. ① Filling the mold

② Putting the box in place

③ Embedded sample Figure 2.10 Embedding a sample in paraffin. • Cool: — On a refrigerating plate, or — In cold water ❺ Storage of blocks

➫ The block comes out of the mold naturally after the completion of cooling. rt

➫ Conservation is indefinite. 37

Preparation of Samples 2.1.5.3

Making sections

❶ Preparing the sections Prepare the sections on a microtome, after the paraffin block has been attached to the sample support arm. The thickness of the sections is maintained at 5 µm.

➫ The sections can be 7 µm thick in the case of delicate structures (e.g., the spleen or the thymus) which are more sensitive to the subsequent treatments.

① Cassette in the sample support arm ② Knife

③ Strip of paraffin sections Figure 2.11 Production of paraffin-embedded sections. ❷ Arranging the sections The sections are arranged on pretreated slides, then are spread hot on a drop of sterile distilled water.

➫ See Appendix A3.

① Paraffin section ② Sterile water ③ Pretreated sterile slide

3

2 1

The spreading takes place on a heating plate maintained at a temperature of 40 to 45°C.

Figure 2.12 Positioning a section on a slide. ➫ This method of spreading sections is the best way of ensuring RNase-free conditions. If the spreading is carried out in a water bath at 40°C, the water must be sterile, and changed regularly. ➫ It is possible to perform PCR or RT-PCR with control on adjacent sections on the same slide, and thus in the same conditions. Figure 2.13 The positioning of three sections on a slide.

≈12 h ❸ Drying in an incubator at 37°C. ❹ Storage of the slides in hermetically rt sealed boxes, with a desiccant. ➲ Following step • Proteolytic pretreatments. 38

➫ It is best to conserve samples in paraffin blocks. ➫ See Section 3.5.

2.1 2.1.5.4

Tissue Samples

Advantages/disadvantages

❑ Advantages • This is the method that gives the best preservation of morphological structures. • Paraffin blocks and slides are easy to store. • Conservation is more or less indefinite. • It is easy to make sections. ❑ Disadvantage ➫ If it can be considered that better amplification compensates for loss of sensitivity, paraffin embedding after fixation by an aldehyde is the method of choice for in situ PCR and RT-PCR.

• Low sensitivity

2.1.6 Nonfixed Frozen Tissue Freezing without fixation involves the cryofixation of samples in their initial state, without any morphological or structural alteration. 2.1.6.1

Freezing protocols

❶ With a coating of O.C.T. mounting medium. • Freezing in liquid nitrogen. ❷ Without a coating • Isopentane cooled in liquid nitrogen • Vapor from liquid nitrogen • Dry ice 2.1.6.2

➫ See Section 2.1.4.2.2. ➫ See Section 2.1.4.2.2.

Frozen sections

Frozen sections are obtained in the same way as fixed frozen tissue. 2.1.6.3

➫ Fresh tissue is, in this case, frozen without cryoprotection.

➫ See Section 2.1.4.3.

Fixation of frozen sections

❶ Cutting frozen sections A set of 10 to 20 sections are cut and kept in the cryostat at −20°C. ❷ Fixation The 10 to 20 slides are fixed 15 min in a tray with buffered 4°°C paraformaldehyde at 4%.

❸ Rinsing in PBS buffer

➫ See Appendix B4.3. ➫ Better results are obtained with fixed sections. There is better adhesion and better conservation of cellular structures, which limits the migration of amplified products.

2 × 10 min 4°°C

39

Preparation of Samples ❹ Dehydration in alcohol baths of increasing strength: 70°, 95°, 100° ❺ Drying ➏ Storage in hermetically sealed boxes, with a desiccant.

5 min per bath rt −20°°C or −80°°C

➲ Following step • Proteolytic pretreatments 2.1.6.4

➫ Dry under a ventilated hood. ➫ Storage has no undesirable effects if the box is opened at room temperature (so that no condensation occurs), and the desiccant is changed each time the box is opened. ➫ See Section 3.5.

Advantages/disadvantages

❑ Advantages • Ease of utilization • Rapidity • Optimal conservation of RNA

➫ Nonfixed samples are, however, more sensitive to temperature differences resulting from handling.

❑ Disadvantage • Morphology is poorly conserved.

2.2

➫ This can be a major drawback with amplification techniques.

CULTURES/CELLULAR SMEARS

In situ PCR and RT-PCR are easy to carry out on cells, whatever their initial presentation, in monolayer cultures, cell suspensions, or smears.

2.2.1

Cell Origin

Handling conditions are strict, and must provide protection against contamination by DNase or RNase. 2.2.1.1

The different possibilities

❶ Using cells in suspension Cells are obtained either from cultures in suspension or after unsticking the cellular layer dissociation by enzymatic treatment and centrifugation.

• Cultured cells on slides or coverslips • Cell pellets

40

➫ RNase-free conditions are ensured by wearing gloves and making sure that the cellular cultures are kept in sterile surroundings.

➫ The original in situ PCR work was done on cells in supension in Eppendorf tubes containing a PCR reaction medium. After amplification, the cells are cytocentrifuged and treated for the detection of products amplified by in situ hybridization. ➫ These are monolayer cultures. ➫ These can be used in the same way as tissue samples.

2.2

Cultures/Cellular Smears

➫ These can be made either by spreading or by cytocentrifugation (cytospins). ➫ Examples are cephalo-rachidian fluid, blood, urine or ascitic fluid.

• Smears on slides ❷ Using a biological fluid • Obtaining cellular smears by spreading or cytocentrifugation on a slide. 2.2.1.2

Diagram of the different steps

❶ Using a monolayer cellular culture

Cell culture

Culture on slide or cover-slide

Cell suspension

Fixation

Fixation

Freezing

Frozen sections

Cryopreservation

Freezing

Paraffin embedding

Paraffin sections

Frozen sections

41

Preparation of Samples ❷ Using a culture in suspension or a biological fluid

Biological fluid

Freezing

Frozen sections

Cell suspension

Cell smear

Cytocentrifugation

Fixation

Fixation

Fixation

Cryopreservation

Paraffin embedding

Freezing

Paraffin sections

Frozen sections

2.2.2 2.2.2.1

Cell Cultures on Slides

Culture

• Using a cell suspension obtained after the removal of cells by an enzymatic treatment: — Trypsin at 0.25% in PBS 5 min — Centrifugation 5 min 300–600 g • On slides treated with silane and placed in a petri dish

• On slides with a culture chamber

❶ Seeding at a concentration of 10 cells/30 ml medium 5

42

➫ See Appendix A3. ➫ The slides that are specially designed for the Perkin-Elmer thermocycler make the task easier. The use of three cover disks and three cover clips per slide makes it possible to test three different conditions. ➫ The slides must be made of glass (due to the high temperature of the denaturation cycle), and thick enough to hold the cover clips firmly.

2.2 ❷ Checking the confluence of the cells ❸ Rinsing in a sterile 0.1 M 2 × 5 min phosphate buffer, or in PBS 2.2.2.2

➫ As for tissue samples, the fixation must be immediate.

PRECIPITATING FIXATIVES

Mixture acetone/ethanol ethanol/methanol ethanol/methanol ethanol/water

2.2.2.2.3

➫ The aim is to eliminate all trace of the culture medium.

Fixation

2.2.2.2.1 CROSS-LINKING FIXATIVES A number of different fixatives can be used, but paraformaldehyde gives the best results. ❶ Paraformaldehyde at 4% in a 0.1 M phosphate buffer ❷ Paraformaldehyde at 2% in a 0.1 M phosphate buffer 2.2.2.2.2

Cultures/Cellular Smears

Concentration v/v 10/90 50/50 80/20 90/10

➫ The results obtained in these fixation conditions are haphazard. Cell morphology is often poorly preserved, and the absence of a signal often indicates no more than the diffusion of PCR products, and/or other cellular components, outside the cells. ➫ Slides fixed in this way can be stored in hermetically sealed boxes at −20°C or −80°C (see Section 3.2.2.2.6).

FIXATION PROTOCOLS

❶ Single fixation The slides are immediately fixed either: • In paraformaldehyde at 4% in a 0.1 M phosphate buffer

• Or in paraformaldehyde at 2% in a 0.1 M phosphate buffer ❷ Double fixation Good results have been obtained with the use of a precipitating fixative, followed by postfixation with paraformaldehyde at 2% in a 0.1 M phosphate buffer. ❸ Rinses Use a 0.1 M phosphate buffer or PBS. ❹ Dehydration Use ethanol baths of increasing strength: 70°, 95°, 100°.

30 min 4°°C

➫ The fixation time is very important: inadequate fixation gives rise to the diffusion of amplified products, whereas excessive fixation retards the penetration of the reagents. Some researchers have, however, had improved results after 10–15 h of fixation.

Several h 4°°C 5 min 4°°C

➫ An example is an alcohol/acetone mixture.

30 min 4°°C 2 × 5 min

➫ This depends on the buffer in which the fixative was diluted.

2 min per bath

➫ These dehydration and drying steps are important, as the presence of water could activate cellular RNase during storage. 43

Preparation of Samples ❺ Drying Dry under a ventilated hood. rt ❻ Storage The slides can be stored at −20°C or −80°C in hermetically sealed boxes, with a desiccant. ➲ Following steps ➫ See Section 3.4. ➫ They can be carried out directly after fixation and rinsing. The slides are then pretreated, dehydrated, dried, and stored at −20°C.

• Permeabilization of the cells • Proteolytic pretreatments

2.2.2.3

Advantages/disadvantages

❑ Advantages • It is easy to make cultures on slides. • No complex handling is required. • The natural spreading of the cells means that their structure is maintained for the purpose of in situ studies.

➫ Almost all the cells are adhesive.

❑ Disadvantage • Some cells are difficult to culture on glass slides.

➫ See above.

2.2.3 Cellular Smears 2.2.3.1

Obtaining samples

• Starting with a cell suspension or a biological fluid, the cells are either: — Spread directly on the 50 µl/slide pretreated slide — Or projected by cytocentrifugation onto pretreated slides. • In both cases the slides are dried. 2.2.3.2

10 min 16,000 g 5 min rt

Fixation protocol

❶ Fixation The slides are fixed in the same way as monolayer cellular cultures. ❷ Dehydration They are dehydrated in alcohol baths of increasing strength. ❸ Storage These are in hermetically sealed boxes. −20°°C

44

➫ To obtain a monolayer spread, the concentration of the suspension must not exceed 6 2 × 10 cells/ml. ➫ Cytospin

➫ See Section 3.2.2.2.

➫ See Section 3.2.2.2.3. ➫ See Section 3.2.2.2.3.

2.2

Cultures/Cellular Smears

➲ Following steps ➫ See Section 3.4.

• Permeabilization • Proteolytic pretreatments 2.2.3.3

Advantages/disadvantages

❑ Advantages • Smears or cytospins are easy to carry out. • No complex handling is required.

➫ Suitable equipment is needed.

❑ Disadvantages • Poor cellular adhesion • Loss of morphology

2.2.4 Cellular Pellets 2.2.4.1

Obtaining samples

Using a cell suspension or a biological fluid, the cells are: • Centrifuged 5 min • Rinsed in a phosphate buffer, or in 5 min a culture medium without serum • Centrifuged 5 min 600–1000 g • Fixed 2.2.4.2

➫ The cells can also be obtained by scraping from the bottom of a culture box. ➫ This is done after elimination of the supernatant.

Fixation protocol

❶ Fixation of the pellet using a buffered fixative, of which the most widely used are formalin and paraformaldehyde at 4% ❷ Rinsing in a buffer solution

15–30 min 4°°C 3 × 5 min

➫ See Appendix B4.3.

➫ It is sometimes necessary to carry out a centrifugation between the different steps to maintain the pellet in place.

The pellet can then be considered as a tissue sample, and treated as such. ➲ Following steps • Freezing, or • Cryoprotection and freezing, or • Paraffin embedding 2.2.4.3

➫ See Section 3.1.3. ➫ See Section 3.1.2.3.2. ➫ See Section 3.1.4.

Advantages/disadvantages

❑ Advantages • The sections adhere well to the slide.

45

Preparation of Samples • Access to the different cellular components does not require permeabilization. • Several different cell types can be processed together. ❑ Disadvantages • The cells lose their characteristic morphology. • The procedure is demanding and painstaking.

46

➫ Internal controls

Chapter 3 Pretreatments

Contents

CONTENTS 3.1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51

3.2

Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

52

3.3

Dewaxing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53

3.3.1 3.3.2

Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53 53

3.4 Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53

3.4.1 3.4.2 3.4.3

Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

54 54 54

3.5 Deproteinization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

54

3.5.1 3.5.2

Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymatic Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.1 Proteinase K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.1.1 Parameters for Use. . . . . . . . . . . . . . . . 3.5.2.1.2 Protocols . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.2 Other Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.2.1 Parameters for Use. . . . . . . . . . . . . . . . 3.5.2.2.2 Protocols . . . . . . . . . . . . . . . . . . . . . . . Chemical Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

54 55 55 55 56 56 57 58 58

3.6 Postfixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

59

3.5.3

3.6.1 3.6.2

Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

59 59

3.7 Optional Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

59

3.7.1

3.7.2

3.7.3

Acetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of Endogenous Enzymes . . . . . . . . . . . . . . . . . . . 3.7.2.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2.2 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2.1.1 Inhibition of Endogenous Alkaline Phosphatases . . . . . . . . . . . . . 3.7.2.1.2 Inhibition of Endogenous Peroxidases . . . . . . . . . . . . . . . . . . . . . Digestion by DNase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.3.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.3.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

59 59 60 60 60 60 60 60 61 61 61

49

Pretreatments 3.8

Dehydration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

62

3.8.1 3.8.2

Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

62 62

Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

62

3.10 Recapitulation of the Consequences of Pretreatments . . . . . . . . . . .

63

3.9

50

3.1

Overview

Whatever the biological material, its nature, or fixation, pretreatment step(s) are indispensable to the permeabilization of cellular and/or tissue structures, and to making the target nucleic acid accessible to primers and enzymes in the amplification medium.

3.1 OVERVIEW Pretreatments comprise four essential steps, which have to be adapted, checked, and optimized for each type of sample. The aim is to achieve a compromise between accessibility to the nucleic acids and an acceptable degree of morphological conservation. It must not be forgotten that the cell acts as a “PCR chamber” in which the amplified products must be maintained. Insufficient pretreatments will lead to false-negative results, while overaggressive pretreatments will produce irreversible alterations in the tissue, thereby giving rise to falsepositive results due to diffusion of the amplified products. • Permeabilization by chemical agents

• Deproteinization by enzymatic or chemical treatment • Postfixation

• Dehydration

• Destruction of targets — DNase

➫ Gloves must be worn; all the products must be “RNase-free”; the solutions must be prepared with DEPC water (see Appendix B1.2); and the equipment must be sterilized. ➫ Pretreatments are generally carried out on fixed tissue or cells just before the PCR/ RT-PCR steps. ➫ They can also be carried out at the same time that frozen sections are being obtained from tissue, whether fixed or not. The slides are then dehydrated and stored at −20°C for amplification. ➫ This is especially important in the case of thick sections derived from paraffin-embedded tissue. ➫ This partially eliminates proteins, and thereby reduces the bridges formed between cell proteins and nucleic acids by aldehyde fixation. ➫ This stabilizes structures that have been weakened by permeabilization and deproteinization, and favors the adhesion of the sections to the slides. ➫ Because deproteinized sections are more easily broken down by RNase, this is an indispensable step, especially for pretreated slides stored at −20°C. ➫ This is essential for thick sections. ➫ The destruction of genomic DNA guarantees specificity. ➫ This serves as a negative control of the in situ PCR reaction.

51

Pretreatments As explained in the previous chapter, regardless of: — The nature of the sample, or — Its preparation

➫ Tissue or cells ➫ Fixation, freezing, embedding, or sections

The aim is to obtain fixed tissue sections, cell smears, or suspensions. It is necessary to distinguish between the different types of pretreatment:

➫ Fixation must be carried out before the pretreatment steps.

— Frozen tissue, fixed either before or after freezing — Paraffin-embedded fixed tissue

➫ Frozen sections brought up to room temperature and rehydrated (see Section 2.1.4) ➫ Sections that have been carefully dewaxed (see Section 2.1.5) ➫ Considered as frozen sections (see Section 2.2) ➫ Permeabilization often sufficient in such cases

— Cellular fractions either on slides or in suspension

3.2 DIAGRAM OF THE DIFFERENT STEPS

52

3.4

Permeabilization

3.3 DEWAXING 3.3.1 Aim Dewaxing consists of eliminating the embedding medium. It also allows the sections to be rehydrated. This step is carried out just before the permeabilizing treatments.

➫ A hydrophobic medium ➫ Sections cannot be stored after dewaxing.

3.3.2 Protocol a. Dewax: • Xylene or a substitute

b. Rehydrate: • Alcohol 100° • Alcohol 95° • Alcohol 70° • NaCl 9‰

3 × 10 min

5 min 5 min 5 min 5 min

➲ Following steps • Permeabilization • Deproteinization

➫ Although the risk of contamination in solvents is minimal, the baths must be changed regularly.

➫ Na ions maintain the nucleic acids in situ. Washing in water must be avoided. +

➫ See Section 3.4. ➫ See Section 3.5.

3.4 PERMEABILIZATION 3.4.1 Aim This step facilitates the penetration of reagents into the cells by the permeabilization of their membranes.

➫ This is a necessary step for thick sections of dense, tough tissue (e.g., muscle, heart, placenta) embedded in paraffin.

Figure 3.1 Aspect of the cell after permeabilization.

53

Pretreatments

3.4.2 Reagents • Digitonin • Detergents:

— Sodium dodecylbenzenesulfonate — Triton X100 — Saponin — Sarcosyl — Nonidet P-40 (NP-40)

➫ For cells on slides or in suspension, the use of detergents provides sufficient permeabilization to make the use of proteolytic enzymes unnecessary. ➫ SDS

3.4.3 Protocols All these detergents are prepared in a PBS or phosphate buffer. a. Dip the slides in one of the following baths: • Digitonin, 0.05% 10 min rt • Triton X-100, 0.1% 5–15 min rt • Saponin, 0.1% 5–15 min rt • Sarcosyl, 0.2% 5–15 min rt • Nonidet P-40, 0.5% 1h at 4°°C b. After rinsing in a PBS or phosphate buffer, the preparations can be deproteinized.

➫ See Appendix B3.4

➫ Room temperature

➫ Used essentially for cell fractions on slides or in suspension

3.5 DEPROTEINIZATION 3.5.1 Aim Incubation in detergent solutions is often insufficient to provide complete permeabilization of cells. In such cases proteolytic digestion is indispensable to the partial elimination of proteins. By breaking down DNA/DNA, and DNAhistones protein cross-linking, it makes nucleic acids more accessible to amplification products. Deproteinization is carried out either: • By enzymatic treatment or • By chemical treatment

54

➫ Proteolysis is clearly “tissue dependent,” and this is a critical step, as the integrity of tissue and cells is to be preserved above all else.

➫ Proteinase K, pronase, pepsin ➫ Acid hydrolysis

3.5

Deproteinization

Figure 3.2 Aspect of the cell after deproteinization.

3.5.2 Enzymatic Treatment 3.5.2.1

Proteinase K

Proteinase K is generally considered a selective protease for proteins associated with nucleic acids. When properly used, it is the enzyme that gives the best result in terms of morphological efficacy and preservation. 3.5.2.1.1

PARAMETERS FOR USE

❶ Concentration The optimal concentration depends on the type of sample (cells, frozen tissue sections, and paraffin-embedded tissue sections). • Cytocentrifuged cells cultivated on slides or in suspension • Frozen sections • Paraffin-embedded sections

➫ Enzymes differ considerably in terms of efficiency, and tests have to be carried out when a different brand, or even a different batch of the same brand, is introduced.

1 µg/ml 1–3 µg/ml 3–10 µg/ml

➫ Moderate digestion (1 µg/ml) gives good results. ➫ The optimal concentration depends on the thickness of the sections.

The concentration also depends on the nature of the tissue. The following values are given as a rough guide, for paraffin-embedded tissue: • Lung

1–3 µg/ml

• Liver, intestine, kidney 3 µg/ml • Brain, pituitary 5 µg/ml • Muscle, heart 10 µg/ml ❷ Dilution buffer Proteinase K is diluted in Tris-HCl/CaCl2 buffer (20 mM Tris, 2 mM CaCl2, pH 7.5). ❸ Temperature Optimal temperature

37°°C

➫ This is one of the most delicate types of tissue.

➫ See Appendix B3.7.2. ➫ This is a specific buffer, as calcium is the cofactor of proteinase K. ➫ The enzyme remains effective at lower temperatures. 55

Pretreatments ❹ Incubation time Incubation time varies.

5 to 30 min

➫ Besides concentration, temperature and incubation time are the two parameters that modulate the action of proteinase K.

a. Rehydrate dewaxed or frozen preparations stored at −20°C:

➫ It is important not to open boxes of sections that have been stored at −20°C until they have reached room temperature. ➫ See Appendix B2.19. ➫ See Appendix B3.4.1. ➫ It is best to use a thermostat-controlled water bath with a shaking mechanism.

3.5.2.1.2

PROTOCOLS

• NaCl 9‰ 5 min • Phosphate buffer 0.1 M 5 min b. Place a tray containing Tris-HCl/CaCl2 buffer in a water bath. Dip the slides in the buffer when it has reached 37°°C. c. Enzymatic treatment: At the last moment, add proteinase K at 37°C, and shake with the slide-tray to homogenize. • Proteinase K 15 min 37°°C d. Stop the reaction by rinsing: • In a Tris-HCl/CaCl2 buffer, and then in a phosphate buffer, or • In a phosphate buffer containing 2 mg/ml of glycine, or • By heat

2 min 5 min 2 × 5 min 2 min 95°°C

e. Postfix:

• 4% paraformaldehyde in a phosphate buffer f. Rinse: • Phosphate buffer • NaCl 9‰

5 min

➫ This is an average time, which must be optimized for each type of sample. ➫ Residual proteolytic activity can deactivate Taq DNA polymerase. ➫ See Appendix B3.7.2. ➫ This change of buffer is meant to inhibit enzymatic action, and to eliminate NH2 groups from the Tris-HCl/CaCl2 buffer. ➫ Glycine is a proteinase K inhibitor. ➫ See Appendix B3.7.3. ➫ Use this for cell cultures on slides and cell smears. ➫ This step stabilizes structures weakened by deproteinization, and is particularly important for frozen tissue. ➫ Favors the maintenance of sections on slides for the amplification steps.

3 × 5 min 2 min

➫ To eliminate traces of fixative ➫ Avoids the precipitation of phosphate in alcohols

Other enzymes can also be used, e.g., pronase or pepsin.

➫ These enzymes were widely used some years ago, but have now been abandoned because they are more difficult to use than proteinase K.

➲ Following step • Postfixation

3.5.2.2

56

Other enzymes

3.5 3.5.2.2.1

Deproteinization

PARAMETERS FOR USE

❶ Concentration As for proteinase K, the concentration depends on the nature of the sample, its fixation, and the thickness of the section. • Pronase — Cytocentrifuged cells 500 µg–1 mg/ml cultivated on slides or in suspension — Frozen sections 500 µg–1 mg/ml — Paraffin-embedded 1–4 mg/ml sections • Pepsin

— Cytocentrifuged cells cultivated on slides or in suspension — Frozen sections — Paraffin-embedded sections ❷ Dilution buffer • Pronase Tris–EDTA (TE) buffer • Pepsin–HCl ❸ Temperature • Pronase

• Pepsin

➫ The activity of this pH-dependent enzyme is maximal at pH 2, and is completely inhibited at pH 8. This is its most useful feature. ➫ Acid hydrolysis causes breaks in DNA.

1 mg/ml

1 mg/ml 1–4 mg/ml

pH 7.6 pH 2–5

rt or 37°°C

➫ Use 10 mM or 50 mM Tris (see Appendix B3.6, TE buffer). ➫ Its activity can be changed by increasing the pH (pepsin solution: 9.5 ml H2O, 0.5 ml 2 N HCl, and 20 mg pepsin). ➫ These enzymes are generally used at room temperature, or, to begin with, at a lower temperature. It is easier to control the “incubation time” factor than the “temperature” factor.

rt or 37°°C

❹ Incubation time • Pronase — Cytocentrifuged cells 1–5 min cultivated on slides or in suspension — Frozen sections 1–30 min — Paraffin-embedded sections 1–30 min • Pepsin — Cytocentrifuged cells cultivated on slides or in suspension — Frozen sections

➫ Tests must be carried out systematically.

15 min

➫ This is a very tricky step.

➫ This incubation time is always longer than for frozen tissue. ➫ These approximate values depend on the temperature and pH of the enzyme.

15–60 min 57

Pretreatments — Paraffin-embedded sections

3.5.2.2.2

15–60 min

PROTOCOLS

1. Pronase a. Rinse the slides in TE buffer. b. Incubate the slides in the pronase solution. c. Rinse in TE–glycine buffer (2 mg/ml). d. Rinse in NaCl 9‰.

5 min 5 min

2. Pepsin a. Rinse the slides in Tris-HCl buffer. b. Incubate the slides in the ~30 min pepsin solution (2 mg/ml in 0.2 N HCl, pH 5).

c. Rinse in Tris-HCl buffer, pH 8.

➫ See Appendix B3.6. ➫ Extemporaneous preparation (see Appendix B2.14.3). ➫ The glycine deactives the pronase. ➫ See Appendix B3.7.3. ➫ The sections can then be dehydrated in alcohol baths of increasing strength, after which they can be dried and stored. ➫ These are the classical conditions. ➫ This solution can be frozen, and will retain its activity for a week. ➫ The temperature and incubation time must be adapted to each type of sample.

5 min

➲ Following step • Postfixation

➫ See Section 3.6

3.5.3 Chemical Treatment Acid hydrolysis is an alternative to proteolytic digestion. Hydrochloric acid partly dissolves the bridges created by aldehyde fixation, and its proteasic action is limited. a. Incubate slides in a solution of hydrochloric acid: • HCl 0.05–0.2 N 10 min rt b. Rinse: • Distilled H2O 2–5 min c. Dehydrate and dry. ➲ Following step • Postfixation

58

➫ This is currently very little used, because enzymatic treatments give better results. ➫ It causes breaks in nucleic acids.

➫ See Section 3.6.

3.7

Optional Steps

3.6 POSTFIXATION 3.6.1 Aim Postfixation improves morphological preservation by stabilizing structures that have been modified by deproteinization. It also restores the adhesion of the sections to the slides for the following steps.

➫ This is an indispensable step for frozen tissue sections and cell smears.

3.6.2 Protocols There are two possibilities: ❶ Paraformaldehyde at 4% in a phosphate buffer • Rinsing in a phosphate buffer • Rinsing in NaCl 9‰ ❷ Alcohol 100°°

5 min 2 × 5 min 2 min 5–10 min

➲ Following steps • Acetylation • Inhibition of phosphatases and endogenous peroxidases • Digestion by DNase • Dehydration and drying

➫ To eliminate the fixative ➫ To avoid phosphate precipitation in the alcohol ➫ Cold alcohol recommended for the postfixation of cell cultures and smears ➫ An optional step (see Section 3.7.1) ➫ According to the revelation system used (see Section 3.7.2) ➫ An optional step (see Section 3.7.3) ➫ An optional step (see Sections 2.1.4.3 and 2.1.5.3)

3.7 OPTIONAL STEPS 3.7.1 Acetylation 3.7.1.1

Aim

Acetylation turns the reactive amine group + (– NH 3 ) of the proteins into substitute amine groups (–NH–CO–CH3), which are neutral. It thus reduces the background noise generated by electrostatic forces. This transformation is carried out by incubating the sections in acetic anhydride (CH3–CO–CH3) in a triethanolamine buffer.

➫ The reaction also affects the nitrogenous bases of the nucleic acids, which can result in an overall reduction in the signal.

59

Pretreatments 3.7.1.2

Protocol

a. Dip the slides in a tray containing the triethanolamine buffer (0.1 M, pH 8), with magnetic shaking. b. Add the acetic anhydride, 0.25% with shaking. c. Incubate.

1 min

d. Rinse: • Phosphate buffer

5 min

• NaCl 9‰ e. Dehydrate.

➫ This is the final concentration. ➫ This compound is viscous, and dissolves only with shaking. ➫ The reaction begins immediately, and is complete in less than a minute. ➫ A single bath is enough to eliminate all trace of triethanolamine.

5 min

3.7.2 Inhibition of Endogenous Enzymes 3.7.2.1

Aim

It is indispensable to eliminate all risk of “false positives” resulting from the revelation of endogenous enzymatic activity.

3.7.2.2 3.7.2.1.1

➫ This step is to be carried out only if a positive signal is observed in the absence of amplification (without Taq DNA polymerase). Very often, variations in temperature inhibit endogenous activity. ➫ This step depends on the enzymatic system used for revelation. ➫ It is carried out either during the pretreatment of the samples or during the detection of the amplified product.

Protocols INHIBITION

OF ENDOGENOUS ALKALINE

PHOSPHATASES

Incubating the slides briefly in a 20% aqueous acetic acid solution is an effective way of destroying hepatic alkaline phosphatases. However, intestinal and placental phosphatases are resistant to this treatment. a. Immerse the slides in a 15 s 20% acetic acid solution. 4°°C b. Rinse in a buffer solution. 3.7.2.1.2 INHIBITION OF ENDOGENOUS PEROXIDASES Hydrogen peroxide (H2O2) destroys endogenous peroxidases. a. Immerse the slides in a 0.1% sodium azide solution containing 0.3% H2O2. b. Rinse in a buffer solution. 60

10 min rt

➫ Levamisol treatment is then necessary just before the detection of the amplified product (see Section 3.7.2.1.1 and Appendix B6.2.1.2).

➫ If not, there is a risk that the nucleic acids may be hydrolyzed.

➫ Hydrogen peroxide can be kept at 4°C. It loses its effectiveness over time, and should be used within 3 months. ➫ See Appendix B6.2.1.3.

3.7

➫ For the detection of mRNA by in situ RT-PCR, or for an in situ PCR control

3.7.3 Digestion by DNase 3.7.3.1

Optional Steps

Aim

DNase treatment destroys DNA (genomic, viral, or plasmid), sparing only target RNA.

➫ This step must not be carried out until after proteolytic digestion, because the DNA must be accessible if the DNase is to be effective. ➫ This step is necessary when RT-PCR uses a pair of primers that are not specific to the RNA sought, as this involves a non-negligible risk of mismatching and nonspecific amplification.

Figure 3.3 Aspect of the cell after DNase.

In most cases, the primers are chosen on either side of an intron or a splicing zone so that the amplification is limited to the sequence being studied.

3.7.3.2

➫ In such cases, this step is superfluous. ➫ See Chapter 5.

Protocol

a. Prepare the DNase solution in a specific buffer (Tris-HCl 40 mM, pH 7.4; MgCl2 6 mM; CaCl2 2 mM).

µl 100 U/µ

b. Cover the sections with 20 to 30 µl of this solution. c. Incubate in a moisture 1h chamber. 37°°C d. Rinse in a DNase buffer. e. Rinse in DEPC-treated sterile water. f. Dehydrate and dry. ➲ Following steps • RT • PCR

➫ The DNase must be of “RNase-free” quality. ➫ For RNase-rich cells, add 1000 U/ml of RNasin + 1 mM dithiothreitol (DTT) to the DNase solution. ➫ Some workers recommend much weaker concentrations of DNase, e.g., 1 U/µl, and an incubation period of up to 18 h. ➫ See Appendix B1.2.

➫ For in situ RT-PCR reaction, see Chapter 4. ➫ See Chapter 5.

61

Pretreatments

3.8 DEHYDRATION 3.8.1 Aim Through the action of alcohols and air, the aqueous medium is eliminated from the section, to: • Avoid any dilution of reagents in subsequent steps • Protect the samples against the action of RNase and DNase

➫ Postfixation by precipitating fixatives

➫ RNase and DNase are inactive in the absence of water.

3.8.2 Protocol a. Dehydrate the preparations in alcohols of increasing strength: • Alcohol 70°, 95°, 100° 2 min/bath b. Dry: • In a vacuum jar >30 min • In air under a hood, >60 min protected against dust ➲ Following steps • Storage at −20°C in hermetically sealed boxes with a desiccant • Reverse transcription • Amplification

➫ Room temperature.

➫ The slides can be stored in this way, pending the PCR or RT-PCR step. ➫ See Chapter 4. ➫ See Chapter 5.

3.9 STORAGE After dehydration, and the evaporation of the alcohol, the slides are stored in hermetically sealed boxes with a desiccant. Storage conditions rt −20°°C or −80°°C Storage time

62

Several months

➫ If the slides are stored at −20 or −80°C, they must be allowed to return to room temperature before the box is opened, to avoid the reactivation of RNase. ➫ In favorable conditions, the storage time can be even longer.

3.10

Recapitulation of the Consequences of Pretreatments

3.10 RECAPITULATION OF THE CONSEQUENCES OF PRETREATMENTS

Dewaxing Permeabilization Deproteinization Postfixation Acetylation Inhibition of endogenous enzymatic activity Digestion by DNase Dehydration

Preservation of Morphology

Accessibility of the Target

Intensity of the Signal

Specificity of the Signal

+++ ++ +++ − −

+ ++ +++ + +

++ + + − ++

++ − − ++

−

−

++

−

++

− ++

− +++

++ ++

− +

++

Control

63

Chapter 4 Reverse Transcription (RT)

Contents

CONTENTS 4.1 The Principle of Reverse Transcription. . . . . . . . . . . . . . . . . . . . . .

69

4.2

Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . .

70

4.3 The Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

70

4.3.1 Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1.1 Poly (T) Primer. . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1.2 Random Primers . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1.3 Specific Primer . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Deoxynucleotide Triphosphates (dNTPs) . . . . . . . . . . . . . . 4.3.3 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3.1 AMV Reverse Transcriptase. . . . . . . . . . . . . . . . . 4.3.3.2 M-MLV Reverse Transcriptase. . . . . . . . . . . . . . . 4.3.3.3 Tth DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . 4.3.3.4 Criteria of Choice . . . . . . . . . . . . . . . . . . . . . . . . .

70 70 71 72 73 74 74 75 75 76

Materials/Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

77

4.4.1

Thermocycler. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1.1 Thermocycler for PCR, Either in Situ or in Liquid-Phase . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1.2 Thermocyclers for in Situ PCR. . . . . . . . . . . . . . . 4.4.1.2.1 Apparatus with Slides Placed Vertically . . . . . . . . . . . . . . . . . 4.4.1.2.2 Apparatus with Slides Placed Horizontally . . . . . . . . . . . . . . Sealing Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2.1 The “Cover Disk/Cover Clip” System . . . . . . . . . 4.4.2.2 The “Easyseal” System . . . . . . . . . . . . . . . . . . . . Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

77

Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

80

4.5.1 4.5.2

81 81 81

4.4

4.4.2

4.4.3 4.5

Reactive Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.2.1 Tissue Sections or Cells on Slides . . . . . . . . . . . . 4.5.2.1.1 Making a Hermetic Incubation Chamber . . . . . . . . . . . . . . 4.5.2.1.2 Incubation Temperature . . . . . . . . . . . 4.5.2.1.3 Duration of Incubation . . . . . . . . . . . . 4.5.2.1.4 Deactivation of the Enzyme . . . . . . . .

77 78 78 78 79 79 79 80

81 85 85 85

67

Reverse Transcription (RT)

4.5.3

68

4.5.2.2 Cells in Suspension . . . . . . . . . . . . . . . . . . . . . . . 4.5.2.2.1 Incubation Temperature . . . . . . . . . . . 4.5.2.2.2 Duration of Incubation . . . . . . . . . . . . 4.5.2.2.3 Deactivation of the Enzyme . . . . . . . . Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.3.1 Tissue Sections or Cells on Slides . . . . . . . . . . . . 4.5.3.2 Cells in Suspension . . . . . . . . . . . . . . . . . . . . . . .

85 86 86 86 86 86 86

4.1

Reverse transcription (RT) is an indispensable step that turns messenger RNA (mRNA) into complementary DNA (cDNA) prior to the DNA polymerase chain reaction (PCR), which specifically amplifies this newly-synthesized DNA. Thus, in situ RT-PCR is the most sensitive method for locating a specific, weakly expressed gene in a cell structure.

The Principle of Reverse Transcription

➫ Gloves must be worn. ➫ RT must be carried out in “RNase free” conditions. All the equipment must be sterilized at 180°C for 3 h, and the water used for the solutions must be sterile, or DEPC-treated (see Appendix B1.2).

4.1 THE PRINCIPLE OF REVERSE TRANSCRIPTION RT requires: • An RNA target matrix • A primer, which may be various types • Triphosphate deoxynucleotides • An enzyme: reverse transcriptase • A reaction medium

➫ ➫ ➫ ➫

See Section 4.5.3.1. See Section 4.5.3.2. See Section 4.5.3.3. See Section 4.5.4.

➫ Single-strand polyadenyl [Poly (A)] RNA. AAAAAA 3’

5’ 1

AAAAAA 5’

3’

➫ Hybridization of the primer: ① Poly (T) (TTTT)

TTTTTTT 2 AAAAAA 5’

3’

3 5’ 3’

5’

RT

5’

AAAAAA 3’ AAAAAA TTTTTTT 5’

3’ TTTTTTT 3’

5’

② Nonspecific primer, either hexamer or nonamer (—) ③ Specific primer (———)

➫ Elongation by a reverse transcriptase (RT) using a poly (T) primer ➫ Obtaining cDNA Figure 4.1 The principle of reverse transcription. 69

Reverse Transcription (RT)

4.2 DIAGRAM OF THE DIFFERENT STEPS

RT

4.3 THE TOOLS

4.3.1 Primers The choice of primer for RT depends on the chosen objective and the experimental approach, but also on the size and the conformation of the mRNA to be transcribed. 4.3.1.1

Poly (T) primer

This is an oligo-(dT) that, by complementarity, hybridizes with the poly (A) end of mRNA. Polymerization starts from this primer, and proceeds in the 5′ → 3′ direction. In this case, all the mRNA is reverse-transcribed into cDNA. It is then possible to amplify two different sequences on the same section, using specific primers.

70

➫ Reverse transcription is either total or specific to the mRNA being sought. ➫ Choices are Poly (T) primer, random or specific primer.

➫ This is usable only with eukaryotes, because prokaryote RNA does not have a poly (A) end. ➫ Multiple labeling is possible. ➫ Several associations are then possible: • Amplification by direct PCR with specific primers and incorporation of a labeled nucleotide and • Indirect PCR using specific primers for the other sequence to be amplified, and detection by hybridization with labeled probes.

4.3

The Tools

➫ mRNA ➫ Hybridization of the poly (T) primer, and reverse transcription

➫ Several different cDNA Figure 4.2 The principle of reverse transcription with a poly (T) primer. ❶ Characteristics • It is generally 18 to 20 mers long. • It must be purified to eliminate small poly (T) primers.

➫ Small poly (T) primers might then be considered as nonspecific primers that can hybridize with repeats of adenosine contained in mRNA.

❷ Primer hybridization temperature • Optimal enzyme temperature is 37 to 40°C. 4.3.1.2

Random primers

These contain six or nine nucleotides, which hybridize in a random way with mRNA, as in “random priming” labeling method, which results in reverse transcription occurring at many points along the transcript.

➫ Hexamers and nonamers are mostly used to retrotranscribe long-strand RNA or sequences with secondary structures.

➫ mRNA ➫ Random hybridization of hexamers or nonamers, and reverse transcription

➫ Numerous cDNA sequences, all different Figure 4.3 The principle of reverse transcription with random primers. ❶ Characteristics • These are generally either hexamers or nonamers.

• Their sequence is randomly chosen.

➫ The probability that such a sequence will encounter a complementary sequence on a 6 strand of RNA is, for a hexamer, 1/4096 (1/4 ) 9 and, for a nonamer, 1/262144 (1/4 ). Thus, the use of hexamers results in larger amounts of cDNA, while use of nonamers results in longer cDNA. ➫ These sequences hybridize with complementary or anti-sense sequences. 71

Reverse Transcription (RT) ❷ Hybridization temperature of random primers Incubation at 25°C for 10 min is indispensable before incubation at the ideal temperature of the enzyme. 4.3.1.3

➫ This facilitates the hybridization process.

Specific primer

In most cases, this is a synthetic oligonucleotide whose sequence is complementary and antisense to the mRNA that is to be amplified.

➫ This is the primer that generally gives the highest degree of specificity.

➫ mRNA ➫ Hybridization of the specific primer, and reverse transcription ➫ cDNA Figure 4.4 The principle of reverse transcription with a specific primer. ❶ Characteristics • Its sequence must be anti-sense and complementary to a unique sequence in the target sequence. 3′

5′ CAG AAT GCC CAG GCT GCG TTC TGC TTC TCA

5′

GAA GCA GAA CGC AGC CTG GGC ATT

5′

➫ cDNA corresponding to the mRNA sequence ➫ Complementary anti-sense sequence

TTA CGG GTC CGT CGC AAG ACG AAG

3′

➫ As regards the cDNA strand with which it is meant to hybridize.

3′

➫ Specific primer for a given sequence Figure 4.5 Determination of a specific primer sequence.

• Its GC content must not exceed 50 to 55%. • It is recommended that there be a G or C base, or, better still, a GC or CG base pair, at the 3′ end, from which the neosynthesis initiates. • It should be between 20 and 30 mers long.

• The 5′ end must not include a sequence that is complementary to a sequence situated at the 3′ end, as this would entail a risk of autoligation. 72

➫ Long sequences (>10 nucleotides) of poly (C), poly (G), or poly (GC) should be avoided. ➫ It is generally agreed that a strong bond at this end improves neosynthesis. ➫ Although an oligonucleotide of more than 18 mers is considered specific, it should be matched against a data bank to make sure that there is no homology with any sequence of the genome. ➫ This is a palindromic sequence.

4.3 ❷ Position The sequence must occur on an exon.

ATG

I1

I2

I3

The Tools

➫ This is often the anti-sense primer of the pair of primers that are necessary to the amplification (see Chapter 5). I4

I = Introns

I4

5′

3′

TATAA

E1

E2

E3

E4

E5

E = Exons, which together make up the cDNA ➫ Anti-sense primer Figure 4.6 The position of the specific primer in the structure of the gene.

❸ Primer hybridization temperature For oligonucleotides, this temperature is based on their sequence, and is calculated as follows: • For oligonucleotides with up to 20 bases Tm = 2 × (A + T) + 4 × (G + C) • For larger oligonucleotides +

Tm = 16.6 log[Na ] + 0.41(% GC) + 81.5 − % mismatches − 67.5/base length − 0.65 (% formamide)

The optimal RT reaction temperature, which is generally at Tm −8°C, has to be confirmed experimentally. If the temperature is too high, the primer hybridizes only partly or not at all. Sequences other than the target RNA may then be converted into cDNA. ❹ Concentration Regardless of primer choice, the final concentration of the primer must be optimized. An addition of 50 pmol of primer is recommended as a starting point for optimization.

➫ This temperature is called the melting temperature (Tm). It corresponds to the formation of 50% of hybrids. ➫ Wallace’s formula ➫ Where A, T, G, and C represent the corresponding numbers of nucleotides ➫ For a given oligonucleotide, the optimal Tm is generally given by the manufacturer. It is worked out by using the “nearest neighbor formula” (Breslauer). ➫ This formula takes into account the saline concentration of the reaction buffer, as well as the GC concentration. ➫ Given the optimal operating temperature of the enzyme (between 37 and 42°C), the hybridization of the primer often takes place at low temperature (less than the suggested Tm). The use of Tth DNA polymerase (see Section 4.3.3.3), which works at 70°C, can then be advantageous.

➫ Final concentration is 1 µM in a 50 µl reaction volume.

4.3.2 Deoxynucleotide Triphosphates (dNTPs) In reverse transcription, four triphosphate nucleotides are used at an equimolar concentration: • • • •

dATP dTTP dCTP dGTP

➫ In the opposite case, the fidelity of the enzyme and the length of the transcribed cDNA can be affected. ➫ Deoxyadenosine ➫ Deoxythymidine ➫ Deoxycytosine ➫ Deoxyguanosine 73

Reverse Transcription (RT)

4.3.3 Enzymes The most frequently used enzymes are: • AMV reverse transcriptase • M-MLV reverse transcriptase • Tth DNA polymerase Given that the manufacturers of these enzymes give information about their particular characteristics, this paragraph will present only their main features and modes of action. 4.3.3.1

AMV reverse transcriptase

❶ Origin AMV reverse transcriptase is extracted from the avian myeloblastosis virus. It is an αβholoenzyme with a molecular weight of 157 kDa. The mature αβ dimer has different enzymatic activities: • RNA-dependent DNA polymerase • DNA-dependent DNA polymerase • RNase H ❷ Quantity supplied The quantity supplied corresponds to a level of activity varying between 250 and 1000 U. The conservation buffer contains 50% glycerol to avoid freezing. ❸ Conservation In order for the enzyme to retain its maximum level of activity, it should in general be stored in aliquots at −20°C.

❹ Principle of action AMV reverse transcriptase is a polymerase DNA that can catalyze the polymerization of cDNA, starting with a single-strand RNA or DNA molecule. ❺ Utilization • Reaction buffer This does not differ much between one supplier and another: only the relative concentrations of the components are different. • Concentration Variable according to the quality of the enzyme. • Temperature Optimal polymerization takes place at a temperature of between 37 and 42°C. 74

➫ Avian myeloblastosis virus ➫ Moloney murine leukemia virus ➫ Thermostable polymerase of Thermus thermophilus ➫ The specifics of the enzyme have to be taken into account in the RT step.

➫ The α subunit is derived from the β subunit by proteolysis.

➫ A unit (U) is defined as the quantity of enzyme which, in 10 min at 37°C, incorporates 1 nmol of insoluble dTTP in an acid medium, starting with an RNA [poly (A)] matrix and an oligo (dT) primer (see Section 4.3.1). ➫ Conservation time is extended by storage at −70°C, and according to some suppliers an enzyme will remain stable for 24 months in such conditions. ➫ To avoid successive warmings, an isothermal refrigerated box should be used. ➫ This reaction requires a primer and Mg ions as cofactors.

2+

➫ Supplied with the enzyme (10X) ➫ For example, Tris–HCl, pH 8.3, 400 mM KCl, 80 mM MgCl2, 10 mM DTT ➫ From 1 to 20 U/µl ➫ Generally 40°C

recommended

temperature:

4.3 4.3.3.2

M-MLV reverse transcriptase

❶ Origin M-MLV reverse transcriptase is a recombinant enzyme. It is cloned from an Escherichia coli strain that expresses the gene of the leukemia virus in the Moloney mouse. Its molecular weight is 71 kDa. It is an RNA- and DNAdependent DNA polymerase. It has been genetically modified so as to eliminate its RNase H activity. ❷ Quantity supplied The quantity supplied corresponds to a degree of activity of between 10,000 and 50,000 U. The conservation buffer contains 50% glycerol to avoid freezing. ❸ Conservation In order for the enzyme to retain its maximum level of activity, it should in general be stored in aliquots at −20°C. ❹ Principle of action M-MLV reverse transcriptase is a DNA polymerase that can catalyze the polymerization of cDNA, starting with a single-strand RNA or DNA molecule. ❺ Utilization • Reaction buffer This does not differ much between one supplier and another: only the relative concentrations of the components are different. • Concentration Variable according to the quality of the enzyme. • Temperature Optimal polymerization takes place at a temperature of 37°C. 4.3.3.3

The Tools

➫ This modification makes it possible to obtain larger fragments when cDNA is being synthesized. A unit (U) is defined as the quantity of enzyme that, in 10 min at 37°C, incorporates 1 nmole of insoluble dTTP in an acid medium, starting with an RNA [poly (A)] matrix and an oligo(dT) primer. ➫ To avoid successive warmings, an isothermal refrigerated box should be used. ➫ This reaction requires a primer and Mg ions as cofactors.

2+

➫ Supplied with the enzyme (5X) ➫ For example, 250 mM Tris–HCl, pH 8.3, 500 mM KCl, 10 mM MgCl2, 40 mM DTT ➫ In general, 50 U/µl

Tth DNA polymerase

❶ Origin Tth DNA polymerase is a recombinant enzyme. It is cloned from the bacterial Thermus thermophilus KTP strain. Its molecular weight is 92 kDa, and it is capable, in the presence of MnCl2 and at a high temperature (74°C), of polymerizing DNA from an RNA matrix. Tth DNA polymerase can also polymerize double-strand DNA nucleotides from a DNA matrix in the presence of MgCl2.

➫ Its half-life is 40 min at 95°C. ➫ This high-temperature reverse transcription activity minimizes the kind of problem that can be caused by the existence of secondary RNA structures, which are unstable at high temperatures. ➫ It has been shown to be effective in synthesizing fragments of up to 12 kb.

75

Reverse Transcription (RT) ❷ Quantity supplied The quantity supplied corresponds to a level of activity varying between 100 and 1000 U. The conservation buffer contains 50% glycerol to avoid freezing. ❸ Conservation In order for the enzyme to retain its maximum level of activity, it should in general be stored in aliquots at −20°C. ❹ Principle of action Tth DNA polymerase has reverse transcriptase activity in the presence of MnCl2, and DNA polymerase activity in the presence of MgCl2. Its synthesis rate is 60 nucleotides per second per enzyme molecule. ❺ Utilization • Reverse transcription buffer

• Amplification buffer

• Concentration Variable according to the quality of the enzyme. • Temperature Polymerization is optimal at 74°C.

4.3.3.4

➫ To avoid successive warmings, an isothermal refrigerated box should be used. ➫ The same enzyme can thus be used for both reverse transcription and amplification by changing the composition of the reactive mixture between the two steps.

➫ Supplied with the enzyme (10X) ➫ For example, 670 mM Tris–HCl, pH 8.8, 166 mM (NH4)2SO4, 25 mM MnCl2 ➫ The MnCl2 solution is supplied separately so that its concentration can be optimized. ➫ Supplied with the enzyme (5X) ➫ For example, 335 mM Tris–HCl, pH 8.8, 83 mM (NH4)2SO4, 50 mM MgCl2, 0.1 mM EDTA, 25% glycerol, 0.1% Tween 20 ➫ The MgCl2 solution is supplied separately so that its concentration can be optimized. ➫ In general, 5 U/µl ➫ The high temperature also ensures a higher degree of primer hybridization specificity and extension reaction.

Criteria of choice

Specificity Origin

AMV

Extracted (Avian myeloblastosis virus) Quantity supplied 250 to 1000 U Storage temperature −20°C or −70°C Stability 24 months Concentration 20 U/µl Optimal activity 40–42°C temperature Cofactor Mg2+ pH buffer 8.3 76

➫ A unit (U) is defined as the quantity of enzyme that, in 30 min at 74°C, incorporates 10 nmol of insoluble dNTPs in an acid medium.

M-MLV Cloned (E. coli) Moloney murine leukemia virus 10,000 to 250,000 U −20°C 24 months 50 U/µl

Tth Cloned (E. coli) Thermus thermophilius 100 to 1000 U −20°C 12 months 5 U/µl

37°C

72–74°C

Mg2+ 8.3

Mn2+ 8.8

4.4

Materials/Reagents/Solutions

4.4 MATERIALS/REAGENTS/SOLUTIONS

4.4.1 Thermocycler These are automatic, programmable machines with a heating block into which tubes or slides can be inserted. They maintain a given temperature for a given period of time. Thus, the temperature and duration of each step can be predetermined, from reverse transcription to the three steps in the PCR cycle. The necessary characteristics of the thermal cycler are: • Speed • Accuracy • Reproducibility

➫ Different types of apparatus are commercially available, some of which are specific for liquid PCR and some for in situ PCR, while others combine the two amplification systems, using interchangeable heating blocks (tubes/ slides). ➫ Each model has both advantages and disadvantages, and rather than make comparisons between them we will simply present one of each type in an objective way.

These qualities determine the yield and efficiency of the amplification process.

4.4.1.1 Thermocycler for PCR, either in situ or in liquid phase This apparatus is used to carry out the reverse transcription and amplification steps on cells in suspension.

➫ The main manufacturers—Applied Biosystems (Perkin-Elmer), Hybaid, MJ Research, and Biometra—produce very similar models whose heating and cooling systems are highly reliable. ➫ Applied Biosystems produces two separate systems, one for liquid phase PCR, the other for in situ PCR, while the other manufacturers have at least one model that combines the two. ➫ This apparatus is dual purpose: it can take either 24 0.2-ml tubes (A) or 16 slides (B). ➫ It can produce a homogeneous temperature (±0.4°C) of 0 to 100°C, and temperature changes take place at 1°C/s for 0.5-ml tubes, and 1.2°C/s for 0.2-ml tubes. ① Tube compartment, which can be replaced by a slide compartment ② Control and programming screen

77

Reverse Transcription (RT) ③ Slide compartment

Figure 4.7 Dual-purpose thermal cycler for liquid phase PCR (A) and in situ PCR (B) (Peltier Thermal Cycler for in situ, PTC-200/ 225, MJ Research). 4.4.1.2

Thermocyclers for in situ PCR

These machines process tissue samples and cells on slides. The two models shown here operate in different ways. 4.4.1.2.1

APPARATUS

WITH

SLIDES

PLACED

VERTICALLY

➫ This thermal cycler can take 10 slides 1.2 ± 0.02 mm thick, placed vertically. It is equipped with the “cover disk/cover clip” system (see Section 4.4.2.1). ➫ It can produce a range of temperatures from 4 to 100°C, and temperature changes take place at 0.67°C/s. ① On/Off ② Slide compartment, which can hold up to 10 slides ③ Blocking bar ④ Control and programming screen Figure 4.8 Thermal cycler for in situ PCR (GeneAmp In Situ PCR system 1000, Perkin Applied Biosystems).

4.4.1.2.2

APPARATUS

HORIZONTALLY

WITH

SLIDES

PLACED

➫ This apparatus can take 20 slides 1 mm thick, placed horizontally. It is equipped with the “Easyseal” system (see Section 4.4.2.2). ➫ It can produce a range of temperatures from 1.5 to 99.9°C, and changes of temperature take place at 0.1 to 0.5°C/s. ① Control and programming screen ② Compartment with a capacity of 20 slides

Figure 4.9 Thermal cycler for in situ PCR (Omnislide System, Hybaid). 78

4.4

Materials/Reagents/Solutions

4.4.2 Sealing Systems To make sure that the reverse transcription and amplification mixtures remain in contact with the section, and that evaporation is avoided during the high-temperature cycles, an incubation chamber needs to be made on the section. The most basic way of doing this is to place a cover clip on a section coated with the reactive medium, and to seal it with a silicone of the rubber cement type. Two systems are commercially available. One of these is specific to Perkin-Elmer Applied Biosystems machines, whereas the other is suitable for all the remaining types of equipment. 4.4.2.1

➫ This system is suitable for the type of PCR apparatus in which the slides are placed horizontally. However, the silicone softens at the high temperatures at which the PCR takes place, and this reduces the effectiveness of the seal. ➫ The “Cover disk/Cover clip” system ➫ The Easyseal system

The “Cover disk/Cover clip” system ① Tissue section on a slide

Side view

② Reactive medium 4

③ Cover disk made of soft plastic ④ Cover clip that seals the cover disk over the slide

3 2

Figure 4.10 The system that is needed for the Perkin Biosystems apparatus, where the slides incubate vertically.

1

4.4.2.2

The “Easyseal” system

This is a system that works for all types of slide, and is eminently suitable for equipment in which the slides incubate horizontally. It forms a sealed chamber around the section. 3

2

① Tissue section on a slide ② Plastic frame whose size is determined by that of the section; its adhesive lower side sticks on the slide, its upper side on the plastic cover slide ③ Plastic cover slide

1

Figure 4.11 The Easyseal system.

79

Reverse Transcription (RT)

4.4.3 Reagents/Solutions • Primer

• Deoxynucleotide triphosphates (dNTPs)

• Dithiothreitol (DTT)

• Sterile water

• Enzyme: reverse transcriptase

®

• Ribonuclease inhibitor (RNasin )

• Buffer (specific to the enzyme)

• MgCl2 solution • MnCl2 solution

➫ See Section 4.5.3.1. ➫ Whatever the type of primer used, the storage concentration is around 10 µM. ➫ Store at −20°C. ➫ This is supplied separately, or in the form of a mix at a concentration of 100 mM. ➫ Store at −20°C. ➫ It is generally supplied with the enzyme at a concentration of 1 mM. ➫ Store at −20°C. ➫ The quality of the water is very important. It must be treated with DEPC (see Appendix B1.2); otherwise, it is very convenient to use 2 ml ampoules of sterile water. ➫ See Section 4.5.3.3. ➫ Activity can vary between 5 and 400 U/µl, according to the type of enzyme used. It is a good idea to check the characteristics of the enzyme, and the protocol suggested by the supplier. ➫ Store at −20°C. ➫ This is a 50-kDa protein that inhibits ribonucleases. This effect is inhibited at temperatures above 50°C. The specific activity is given by the supplier (average: 40 U/µl). ➫ Its composition is optimized by the supplier. It comes in 5× or 10× form. It has a high 2+ MgCl2 concentration, and indeed Mg influences the activity of the enzyme: an excess reduces its usual reaction, whereas a shortage reduces its reactive yield. For this reason, the MgCl2 solution is sometimes supplied separately, and particularly in the case of Tth DNA polymerase. ➫ Store at −20°C. ➫ The MgCl2 concentration needs to be optimized. ➫ Use only if Tth DNA polymerase is used.

4.5 PROTOCOL The reverse transcription step is carried out after the particular pretreatments needed by each sample:

80

➫ See Chapter 4.

4.5

• Frozen tissue or paraffin-embedded sections • Cytocentrifuged cells, or cells cultivated on slides

Protocol

➫ The wearing of gloves and RNase free conditions are obligatory. The quantity and quality of the RNA matrix are decisive for the yield (i.e., the quantity of DNA finally obtained).

• Cells in suspension, whether from cultures, biological fluid, or even the enzymatic dissociation of tissue

4.5.1 Reactive Medium The mixture is prepared in sterile 0.2 ml microtubes specially designed for PCR. It contains: • • • • • • •

Reverse transcription buffer DTT dNTPs in equimolar concentration Ribonuclease inhibitor, RNasin Anti-sense, poly (T), or hexamer primer Sterile water Reverse transcriptase: — AMV — M-MLV — Tth • MgCl2 solution

• MnCl2 solution

➫ Final concentration: 1X ➫ Final concentration: 10 mM ➫ Final concentration: 0.5 mM µl ➫ Final concentration: 1 U/µ µ ➫ Final concentration: 1 M ➫ To a final volume of 100 µ l µl ➫ Average final concentration: 10 U/µ A recommendation on the number of effective units per reaction is generally given by the supplier. ➫ If this is not included in the RT buffer, it is supplied separately at a given concentration. It is then necessary to test different concentrations of between 0.5 and 4 mM, according to the supplier’s recommendations. ➫ Attention: Tth DNA polymerase displays reverse transcriptase activity only in the pres2+ ence of Mn ions. ➫ Final concentration: 1 mM

4.5.2 Reverse Transcription 4.5.2.1 4.5.2.1.1

Tissue sections or cells on slides MAKING

A

HERMETIC

INCUBATION

➫ An indispensable procedure

CHAMBER

❶ The “Cover disk/cover clip” system

81

Reverse Transcription (RT) ① Cover disk ② Cover clip with grips facing forward and fully open ③ Magnetic slot that allows the cover disk/ cover clip assembly to be held in position ④ Red indicator light that shows that the heating block is in operation ⑤ Green indicator light that shows that the temperature has reached 70°C (the heating system is not used for the reverse transcription step, but to carry out a hot start before the amplification step; see Section 4.5.2) ⑥ Heating platform on which the slide is placed; after the cover clip has been put in place, the slide can move to the left between two runners ⑦ Blocking system ⑧ Compression arm knob Figure 4.12 Sealing system assembly tool (PE Applied Biosystems): open position. ⑦ Compression arm knob ⑧ Blocking system in the closed position ⑨ Sliding handles (in the direction of the arrows); this action pushes the sliding grips of the Ampli Cover Clip under the slide, anchoring the Ampli Cover Disk to the slide.

Figure 4.13 Sealing system assembly tool (PE Applied Biosystems): closed position. a. Place the slide on the platform with the first section facing the alignment marks.

➫ The apparatus must not be switched on at this stage, as a temperature of 70°°C would inhibit the activity of the enzyme.

Figure 4.14 Putting the slide in position. b. Place the cover disk in the cover clip, and attach the combination to the magnetic slot on the compression arm. c. Place 20 to 30 µl of the reaction mixture on the sections or cells. d. Seal. 82

4.5

Protocol

① Front view ② Side view, with the back of the slide uppermost

Figure 4.15 Sealed incubation chambers. e. Incubate in the thermal cycler. ① Bar in loading position, which means that the slides can be inserted easily. This type of apparatus can hold ten slides.

1

4 5 2 6 1

7

8

3

Figure 4.16 Arrangement of the slides in the thermal cycler (PE Applied Biosystems). ① Back of the slide against the heating block ②, ③ Clips in the closed position ④ Heating block ⑤ Cover clip ⑥ Cover disk ⑦ Incubation chamber ⑧ Metal strip retaining the slide against the heating block Figure 4.17 Side view of the slide in its compartment.

❷ The Easyseal system

➫ For all the other types of apparatus in which incubation takes place in a horizontal position.

a. Wipe the slide as carefully as possible around the section so that the adhesive frame sticks firmly to the slide. ➫ The adhesive frame (double-sided adhesive) exists in several sizes, and the choice will depend on the size of the section.

Figure 4.18 Placing the adhesive frame on the slide. 83

Reverse Transcription (RT) b. Remove the paper top liner from the frame.

Figure 4.19 Removal of the paper top liner. c. Place 30 to 50 µl of the reaction mixture on the section. ➫ The adhesive frame marks out the edges of the incubation chamber.

Figure 4.20 Placing the reverse transcription medium on the section. d. Carefully lower the polyester cover over the frame starting at the end, where the reagent has been pipetted. This plastic cover sticks to the adhesive frame. ➫ A regular movement in the direction of the arrow. This procedure avoids the formation of bubbles.

Figure 4.21 Sealing the plastic cover. e. Incubate in the thermal cycler. ① Filling the two trays that make up the damp chamber with sterile water

1

3

② Placing the slides in the holder, which can take up to 20 slides ③ Slide-blocking system

2

84

4.5

Protocol

➃ Locking down the cover before switching on the machine, which will already have been programmed

4

Figure 4.22 Placing the slides in the Hybaid Omnislide apparatus. 4.5.2.1.2 INCUBATION TEMPERATURE This does not depend on the choice of primer.

This temperature varies according to the choice of enzyme: ❶ For AMV reverse transcriptase, a temperature of between 37 and 42°°C is recommended. ❷ The temperature for the optimal activity of M-MLV reverse transcriptase is 37°°C. ❸ The temperature for the optimal activity of Tth DNA polymerase is 74°°C.

4.5.2.1.3 DURATION OF INCUBATION This is constant, whatever the choice of enzyme.

1h

4.5.2.1.4 DEACTIVATION OF THE ENZYME The simplest method is thermal 2 min deactivation. 94°°C 4.5.2.2

➫ With random primers, however, i.e., hexamers or nonamers, incubation must be carried out for 10 min at 25°C prior to incubation at the temperature that works best for the enzyme. ➫ It is advisable, in all cases, to use the temperature suggested by the manufacturer of the enzyme. ➫ The temperature that most commonly produces optimal activity is 40°C. ➫ For some enzymes, this temperature can be as high as 42°C. ➫ The advantage with this temperature is that it is generally the same as the hybridization temperature of the primer.

➫ This is the temperature necessary for the destruction of the enzyme.

Cells in suspension

The cells are pretreated and centrifuged. The 6 concentration of the cell pellet is 2 × 10 cells/ml.

➫ See Chapter 3.

a. Prepare the reaction medium in a sterile microtube, without adding the amount of sterile water necessary to the final dilution. b. Place the cell pellet in suspension in the calculated amount of sterile water. c. Add the reactive medium, and homogenize by careful pipetting. µl d. Add the reverse transcriptase. 200 U/µ e. Homogenize further by careful pipetting. f. Carry out the incubation in a liquid-phase PCR apparatus.

➫ See Section 4.5.2.

85

Reverse Transcription (RT) 4.5.2.2.1 INCUBATION TEMPERATURE The incubation temperature depends on the enzyme, and not the primer. 4.5.2.2.2 DURATION OF INCUBATION It is constant, whatever the choice of enzyme.

➫ See Section 4.5.2.1.2.

1h

4.5.2.2.3 DEACTIVATION OF THE ENZYME a. Add a heating step to the RT 2 min procedure. 94°°C b. Lower the temperature. 4°°C

➫ This temperature is necessary for the destruction of the enzyme. ➫ This temperature allows the slides to be left in the PCR apparatus without risk.

4.5.3 Washing 4.5.3.1

Tissue sections or cells on slides

a. Remove the combination of cover disk and cover clip, the Easyseal system, or simply the sealed cover clip, as the case may be. b. Wash in a 0.1 M sterile phosphate buffer. c. Rinse in sterile 9‰ NaCl.

5 min 2 min

d. Dehydrate in alcohol baths 2 min of increasing concentration: per bath 95°, 100°. e. Allow the slides to dry under the hood. ➲ Following step • Amplification by PCR 4.5.3.2

➫ This prevents the phosphate from precipitating in the alcohol baths and producing white streaks.

➫ The slides can be stored in a box with a desiccant at room temperature (for rapid utilization) or −20°C (for longer conservation). ➫ See Chapter 5.

Cells in suspension

a. Centrifuge.

2 min 1500 g b. Eliminate all the supernatant, and put the cells back in suspension in about 200 µl of PBS. c. Centrifuge. 2 min 1500 g d. Eliminate the supernatant, and put the cells back in suspension in 100 µl of PBS. ➲ Following step • Amplification by PCR

86

➫ This is a delicate step, during which the tissue section or cells risk being damaged due to a “suction” effect. The removal should be carried out as gently as possible. ➫ This step is carried out in a tray.

➫ In most cases, the cell pellet is clearly visible.

➫ The cells are then directly usable for PCR. They can, however, be frozen and stored at −80°C in aliquots of 10 or 20 µl. ➫ See Chapter 5.

Chapter 5 Polymerase Chain Reaction (PCR)

Contents

CONTENTS 5.1

Principles of In Situ PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . .

91

5.1.1 5.1.2 5.1.3

Amplification of Double-Stranded DNA . . . . . . . . . . . . . . . Amplification of Reverse-Transcribed cDNA . . . . . . . . . . . Exponential Amplification . . . . . . . . . . . . . . . . . . . . . . . . . .

92 93 94

5.2

Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . .

95

5.3

Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

96

5.3.1

Deoxynucleotide Triphosphates (dNTP) . . . . . . . . . . . . . . . 5.3.1.1 Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1.2 Working Concentration. . . . . . . . . . . . . . . . . . . . . 5.3.1.3 Labeled Deoxynucleotides . . . . . . . . . . . . . . . . . . 5.3.1.3.1 Antigenic Labels . . . . . . . . . . . . . . . . . 35 33 5.3.1.3.2 S and P Radioactive Labels . . . . . . 5.3.2 Primers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2.1 Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2.2 Position on the Gene Structure . . . . . . . . . . . . . . . 5.3.2.3 Primer Hybridization Temperature . . . . . . . . . . . . 5.3.2.4 Conservation and Storage . . . . . . . . . . . . . . . . . . . 5.3.2.5 Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2.6 Validation of the Primer Pair by Liquid-Phase PCR. . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2.7 Labeled Primers . . . . . . . . . . . . . . . . . . . . . . . . . . 35 33 5.3.2.7.1 Radioactive Labels ( S or P) . . . . . . 5.3.2.7.2 Advantages/Disadvantages . . . . . . . . . 5.3.2.7.3 Nonradioactive Labels. . . . . . . . . . . . . 5.3.2.7.4 Advantages/Disadvantages . . . . . . . . . 5.3.3 Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3.1 Taq DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . 5.3.3.2 Other Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . ® ® 5.3.3.2.1 Pfu and Tgo DNA Polymerase . . . . ® 5.3.3.2.2 Extrapol DNA Polymerase . . . . . . . . 5.3.3.2.3 Tth DNA Polymerase . . . . . . . . . . . . . 5.3.3.3 Criteria of Choice . . . . . . . . . . . . . . . . . . . . . . . . .

96 96 96 96 96 99 99 100 100 100 101 101

Equipment/Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . .

109

5.4.1 5.4.2

109 110 110 110 111

5.4

5.4.3

Thermocyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sealing Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2.1 The Different Systems . . . . . . . . . . . . . . . . . . . . . 5.4.2.2 Sealing Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . Reagents/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

101 102 102 103 104 104 104 105 107 107 108 108 109

89

Polymerase Chain Reaction (PCR) 5.5

Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112

5.5.1

112 112 113 114 114 114 115 115 116 117 117

5.5.2

5.5.3 5.5.4 5.5.5 5.5.6 5.5.7

90

Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.1.1 Direct PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.1.2 Indirect PCR/RT-PCR . . . . . . . . . . . . . . . . . . . . . . The Hot Start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.2.1 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.2.2 Avoiding Hot Starts. . . . . . . . . . . . . . . . . . . . . . . . The Amplification Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . Number of Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Programming the Thermocycler . . . . . . . . . . . . . . . . . . . . . The Particular Case of Cells in Suspension . . . . . . . . . . . . . Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.7.1 Washing and Postfixation of Sections or Cells on Slides . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.7.2 Washing Cells in Suspension . . . . . . . . . . . . . . . .

117 118

5.1

Since 1985, genic amplification techniques of the PCR (polymerization chain reaction) type have made it possible to exponentially amplify, in vitro, specific nucleotide sequences of which there may be as little as a single copy. PCR and RT-PCR can thus be used to neosynthesize a large number of copies of a specific DNA or RNA sequence. In situ PCR and RT-PCR combine the advantages of genic amplification with the intracellular localization of nucleotide sequences visualized by in situ hybridization. It is thus a highly sensitive method, which can be used with paraffinembedded sections, frozen-tissue sections, cells that have been centrifuged or cultured on slides, or cells in suspension.

Principles of In Situ PCR and RT-PCR

➫ Gloves must be worn. ➫ RNase-free conditions are very important. All the equipment must be sterilized at 180°C for 3 h, and water used for solutions must be sterile or DEPC-treated (see Appendix B1.2).

5.1 PRINCIPLES OF IN SITU PCR AND RT-PCR Sequences of interest are amplified within cells in the presence of: • Specific short primers (synthetic oligonucleotides) which flank the target sequence • dNTPs • A thermostable enzyme, e.g., Taq DNA polymerase • In a reaction environment (buffer, MgCl2, etc.)

➫ See Section 5.3.2. ➫ See Section 5.3.1. ➫ See Section 5.3.3.

An amplification cycle comprises three key steps: • Denaturation of the target sequences

• Hybridization of the primers

• Extension of the primers by copying the DNA template • Each cycle is repeated 20 to 30 times.

➫ See Chapter 1. ➫ This step is accomplished by breaking hydrogen bonds at high temperatures (94 to 100°C). ➫ This step has to be optimized according to the particular characteristics of the primers. The hybridization temperature can be anywhere between 50 and 70°C. ➫ This is due to the DNA polymerase activity of the enzyme and the dNTPs added to the reaction medium. ➫ The number of cycles has to be optimized according to the nature of the tissue and the target sequence. 91

Polymerase Chain Reaction (PCR)

5.1.1 Amplification of Double-Stranded DNA ➫ Endogenous DNA

1st cycle

3'

5'

5'

3'

① Denaturation at 94°C to separate out and linearize the two DNA strands

1

5'

3'

3'

5' 2

5'

3' 3'

5'

3'

5'

3'

5' 3

5' 3'

② Hybridization of primers on the complementary sequence of each DNA strand; this step is generally carried out at a temperature of 50–60°C

3' 5'

5'

5' 4

③ Extension: Taq DNA polymerase attaches to the 3′ end, and adds the dNTP present in the medium, in the 3′ → 5′ direction, using the complementary strand as a model. Neosynthesis begins when the optimal reaction temperature of the enzyme is reached (i.e., 72–74°C, depending on the manufacturer).

3' 5' 5'

3'

3'

3'

5'

④ By the end of the first cycle, two double strands of DNA have been obtained.

5'

2nd cycle 5'

3'

3'

① Denaturation

5' 5'

3'

3'

5'

② Hybridization

2 3

92

③ Extension

5.1 5'

Principles of In Situ PCR and RT-PCR

3' 5' 5'

3'

5' 3'

5' 5' 5' 3'

5'

④ By the end of the second cycle, four double strands of DNA have been obtained. The amplification is exponential.

4

5' 3'

3'

5' 3' 5'

5' 3'

5' 3'

By the end of the second cycle, two copies of the sequence of interest have been obtained. 3'

5'

5'

3'

5'

Figure 5.1 The PCR principle, the first and second amplification cycles.

5.1.2 Amplification of Reverse-Transcribed cDNA 1st cycle 3' 5'

3' 1 5'

3'

3'

5' 2

3'

5' 5'

3' 3

3'

5' 5'

3' 4

5'

3'

3'

5'

➫ RNA ➫ cDNA obtained by reverse transcription from an anti-sense primer ① Denaturation at 94°C: the cDNA strand separates out from the RNA strand and linearizes ② Hybridization of the sense primer on the complementary cDNA sequence at 50–60°C ③ Extension by Taq DNA polymerase, which can attach only to the 3′ end of a DNA sequence ④ It is thus only at the end of the first cycle that the double-stranded form reappears (see Figure 6.1). After the denaturation that takes place during the second cycle, the two primers can position themselves, and the sequence of interest will be synthesized in the same way by the end of this cycle. Figure 5.2 PCR, using cDNA obtained by reverse transcription of the target RNA.

93

Polymerase Chain Reaction (PCR)

5.1.3 Exponential Amplification With each cycle the number of DNA strands doubles, so that the repetition of the cycles leads to the exponential amplification of a specific nucleotide sequence according to the theoretical formula: n number of copies = 2 where n represents the number of cycles. 3′

5′

5′

3′ 5′

5′ 3′

5′

3′ 5′ 3′

5′

3′

5′ 3′

Second cycle: By the end of this cycle, two fragments of the required size have been obtained.

5′

5′

3′

5′

5′

First cycle: No fragment of the desired size has been synthesized.

3′

3′

3′

➫ The yield is never 100%; for liquid-phase PCR it is closer to 70%. It is difficult to define in situ. ➫ Target DNA, with the sequence of interest

3′

5′

➫ In other words, 20 amplification cycles pro20 duce 2 copies.

3′

3′

3′

5′

5′

5′

Third cycle: Two double strands of the required size have now been synthesized.

5′ 3′ 5′ 3′

3′

3′

3′

3′

5′

5′

5′

5′

3′

Fourth cycle: Eight double strands have now been synthesized. Figure 5.3 Exponential amplification of a sequence of interest during the first four PCR cycles.

94

5.2

Diagram of the Different Steps

5.2 DIAGRAM OF THE DIFFERENT STEPS

Frozen sections Cell smears

Paraffin sections

Cell suspensions

Pretreatments

RNA

Reverse transcription

Washing

Amplification

DNA

Amplification

Washing

Fixation of amplified product

Washing

Fixation of amplified product

95

Polymerase Chain Reaction (PCR)

5.3 TOOLS 5.3.1 Deoxynucleotide Triphosphates (dNTP) 5.3.1.1

Characteristics

These are generally supplied either: • In liquid form, at pH 7, in ultrapure water or in lyophilized form or • Separately (dATP, dCTP, dGTP, dTTP), or in equimolar mixtures 5.3.1.2

Working concentration

The usual concentration is between 50 and 250 µM; an equimolar concentration ensures the fidelity of the enzyme.

5.3.1.3

➫ An excess of dNTP can bring about the amplification of nucleotides that have been poorly, or not at all, incorporated, thus reducing the fidelity of the enzyme.

Labeled deoxynucleotides

Numerous authors have advised against the direct incorporation of labeled nucleotides during the amplification step. The number of nonspecific incorporations, and thus false positive results, can be significant. These nucleotides are nonetheless used to label primers. 5.3.1.3.1 ANTIGENIC LABELS These are nucleotide triphosphates with nitrogenous bases onto which antigenic molecules have been grafted by chemical coupling. The most commonly used reagents are biotin, digoxigenin, and fluorescein. ❶ Biotin Also known as vitamin H, its molar mass is 244, and it has a particular affinity for:

• Avidin (or extra-avidin) • Streptavidin

96

➫ Their degree of purity, as given by the manufacturers, is generally >98%. ➫ In liquid form, dNTPs are supplied at a concentration of 100 mM.

➫ For direct in situ PCR

➫ See Section 5.3.2.7.

➫ These labels are grafted by substitution of a thymidine –CH3 radical, which turns them into deoxyuracil (dUTP). −1

➫ Affinity: 10 M ➫ Note: Many types of tissue, such as the liver, the intestine, and the endometrium have large amounts of endogenous biotin, which can cause interference during the detection of biotinylated amplified products. ➫ Glycoprotein extracted from egg white ➫ Glycoprotein extracted from a Streptomyces avidii culture medium 15

5.3 It can also be detected by an anti-biotin antibody. Labels: These proteins, and the antibody, can be conjugated with:

Tools

➫ Polyclonal or monoclonal

➫ Peroxidase or alkaline phosphatase ➫ Fluorescein, Rhodamine, Cyanine 3, Alexa, etc. ➫ Visualization of the amplified product by electron microscopy; see Section 8.13.

• An enzyme or • A fluorochrome or • Colloidal gold O O O

C O

O

O

O- P

P

P

HN O

OH OH OH CH 2 5′

O

3

4

5

C 2 1 6 CH O N

4′ 3′

N H

H N O

O N H

HN

NH

H

H S

1′ 2′

OH

Uracil nucleotide

➫ The biotin molecule ➫ X-dUTP biotin, where X represents the number of carbon atoms (between 6 and 21) that separate the biotin molecule from the base ➫ The most commonly used labeled nucleotide is 11-dUTP biotin.

R

Attached carbon chain

biotine

Figure 5.4 A biotinylated nucleotide.

❑ Advantages • Four fixation sites for avidin or streptavidin • Biotin-labeled nucleotides can be incorporated directly during the amplification process • Low molecular masss

➫ The efficiency of the detection process ➫ Notably for the detection of viral DNA, e.g., the human papilloma virus (HPV) (see Chapter 11; Figures 11.1 to 11.3) ➫ Small steric hindrance, which favors the incorporation of the labeled nucleotide

❑ Disadvantages • Its utilization is limited by the fact that many types of tissue contain endogenous biotin. • The specificity of anti-biotin sera must be checked. ❷ Digoxigenin Extracted from digitalis (Digitalis purpurea or D. lanata). Its molar mass is 300 to 400. Revelation by an anti-digoxigenin IgG, which can be conjugated to: • An enzyme • A fluorochrome • Colloidal gold

➫ The use of blocking agents (see Appendix B6.2.1) ➫ Inhibition by preincubation with a solution of biotin (homologous antigen) ➫ An antigenic molecule, which does not exist in animal tissue ➫ Either polyclonal or monoclonal ➫ Alkaline phosphatase or peroxidase ➫ Fluorescein, Rhodamine, Cyanine 3, Alexa, etc. ➫ Visualization of the amplified product by electron microscopy (see Section 8.13)

97

Polymerase Chain Reaction (PCR) ➫ The digoxigenin molecule

OH O

CH 3

➫ X-dUTP digoxigenin, where X represents the number of carbon atoms (between 6 and 21) that separate the biotin molecule from the base ➫ The most commonly used labeled nucleotide is 11-dUTP digoxigenin.

OH CH3 O HN

O O O P

P

P O

OH OH OH CH 2 5′

C 4

H O N (CH2)5

5

O C 2 1 6 CH O N 4′ 1′ 3′ 2′

O O

N H

O

O

3

R

OH

Figure 5.5 Nucleotide coupled to digoxigenin.

Attached carbon chain

Uracil nucleotide

❑ Advantage • Detection is specific to the animal kingdom.

➫ Animal tissue does not contain any endogenous digoxigenin.

❑ Disadvantages • Structure of digoxigenin similar to that of steroids • Toxicity • A high molecular masss ❸ Fluorescein This is a fluorochrome which, when stimulated by a photon, emits another photon of higher wavelength. This property is responsible for the fluorescence produced by ultraviolet radiation.

➫ Nonspecific bonds can be formed with some receptors. ➫ This must not be inhaled. ➫ This limits its incorporation. ➫ Mw: 332 ➫ Not its most useful property; this compound is generally considered an antigen that can be detected by an antifluorescein antibody.

OH

➫ The fluorescein molecule C

O+

O C HN 4 3 5

O O O

6 CH O- P P P O O C 1 N O OH OH OH CH2 1′ 4′ 5′ 3′ 2′ 2

OH

Uracil nucleotide

98

O H N N N

O HO O

➫ X-dUTP fluorescein, where X represents the number of carbon atoms (generally 11 or 16) that separate the fluorescein molecule from the base

R

Attached carbon chain

Figure 5.6 Nucleotide coupled with fluorescein.

5.3 This labeled nucleotide can be incorporated directly during the amplification process, but it is more generally used to label primers during their synthesis. Fluorescein, like other fluorochromes, is especially used in PCR and RT-PCR techniques with cells in suspension.

Tools

➫ In both cases, the amplification product can be visualized directly by fluorescence microscopy. Its detection by an immunocytochemical reaction amplifies the signal. ➫ Flow cytometry makes it possible to detect rare positive cells.

❑ Advantages • The possibility of visualizing the amplified product directly • A rapid method • The possibility of amplifying the signal by an immunohistochemical reaction

➫ This is possible providing the target sequence is sufficiently strongly expressed. ➫ It allows different pairs of primers to be tested rapidly. ➫ This takes longer, but is also more sensitive.

❑ Disadvantages • The disadvantages generally associated with fluorochromes • Specific equipment required for visualization • Fluorescence poorly conserved over time

➫ Despite storage of the slides at 4°C, and the antifading substance that is added to current embedding media

• Low sensitivity in direct visualization 35

33

5.3.1.3.2 S AND P RADIOACTIVE LABELS Radioactive nucleotides are not used in direct incorporation, but may be used to label directPCR primers.

➫ As well as a lack of specificity, there is a high risk of contamination. ➫ See Section 5.3.2.7.

5.3.2 Primers The primers are a pair of synthetic oligonucleotides whose sequence is complementary and anti-sense to each of the DNA strands. Although the particular characteristics of each primer are identical to those of the specific anti-sense primer used for the reverse transcription, there are some rules that have to be respected regarding their position on the structure of the gene. A pair of primers is generally enough to produce amplification, although another pair of primers situated on the sequence amplified by the first pair of primers may also be used.

➫ cDNA produced by reverse transcription is double-stranded after the first PCR cycle. ➫ See Section 4.3.1.

➫ This method is known as nested PCR. It makes possible a further amplification of the product after its amplification by the first primers, thus increasing sensitivity and specificity.

99

Polymerase Chain Reaction (PCR) 5.3.2.1

Characteristics

• A primer consists of a sequence of 20 to 22 mers, containing 50 to 55% GC, which is anti-sense and complementary to a single sequence of the target DNA. • Sequences that could give rise to secondary structures should be avoided. • The 3′ ends of the primers must not be complementary if the formation of dimer is to be avoided. 5.3.2.2

➫ The degree of homology with other sequences that might be expressed in the tissue under consideration has to be checked against data banks. False-positive results are usually caused by primer mismatches (see Section 9.4). ➫ Autoligation phenomena ➫ Palindromic sequences

Position on the gene structure

• The primers must enclose the sequence of interest. • In situ PCR necessitates a pair of primers capable of generating fragments >400 bp, although PCR efficiency is higher, and especially in the liquid phase, if primers that generate 150 to 300 bp fragments are used. • To avoid the amplification of genomic DNA, it is best to choose sequences from different exons that enclose one or more introns.

A gene is defined by its exons, which make up the totality of the protein-coding region. ATG

I1

I2

I3

I4

I4

5′ TATAA

3′ E1

E3

E2 5′

E5

E4

3′

3′

➫ Each 3′ end terminates with a primer. ➫ Purpose is to limit problems of diffusion. Smaller fragments could diffuse through the nuclear and cytoplasmic membranes, even after permeabilization during the pretreatment steps. ➫ If the primers that enclose the sequence of interest in the cDNA also enclose one or more introns in the genomic DNA, the sequence to be amplified will be much too long, and the extension phase too short, to allow this synthesis to take place. ➫ cDNA is made up of the exons as a whole. I = Introns E = Exons ➫ Sense and anti-sense

5′

Figure 5.7 The positions of the primers on the structure of the gene. 5.3.2.3

Primer hybridization temperature

• The hybridization temperature will be that of the primer with the lowest Tm. • This is generally around 54°C

100

➫ The ideal situation is when the two primers hybridize at the same temperature. ➫ Tm = melting temperature. ➫ A higher hybridization temperature would not be a problem: the smaller the difference between the hybridization temperature and the extension temperature, the better the thermocycler performs.

5.3 Example of a calculation for a 22-mer oligonucleotide with 60% GC in 50 mM KCl: Tm = 81.5 + 16.6 × (log[KCl]) + 0.41 (% GC) − 675/22 Tm = 81.5 + 16.6 × (log[0.05]) + 0.41 (60) − 675/22 Tm = 81.5 + 16.6 × (−1.30) + 24.60 − 30.68 Tm = 53.84°C 5.3.2.4

They are reconstituted in DEPC-treated water. Storage is at −20°C.

➫ But the addition of both primers at a concentration of 50 pmoles is a good starting point. ➫ Final concentration: 1 µM, for a reaction volume of 50 µl

Validation of the primer pair by liquid-phase PCR

Before performing in situ PCR or RT-PCR, the primers should be tested by liquid-phase PCR or RT-PCR on control cDNA or RNA extracted from tissue or cells known to express the gene being sought. The amplification product is analyzed by electrophoresis on agarose gel to find out whether: • Amplification has occurred • The amplified product does or does not correspond to the expected fragment • The amplified product does or does not correspond to the gene being studied

1

2

3

4

5

1000 bp 500 bp

➫ This guarantees their stability during transport, and allows the user to reconstitute them at the chosen concentration (e.g., 10 µM). ➫ Or in TE buffer.

Concentration

Primers must be at an equimolar concentration in the reaction mixture. This concentration will need to be optimized.

5.3.2.6

➫ According to Breslauer’s formula (see Section 3.1.3).

Conservation and storage

Oligonucleotides are generally supplied in lyophilized form.

5.3.2.5

Tools

427 bp

➫ It is necessary to identify the gene of interest by liquid-phase RT-PCR in RNA extracted from the tissue being studied, prior to determining its cellular location by in situ PCR. ➫ See Appendix A4. ➫ Validation of the methods, hybridization temperatures, and products ➫ Verification of size by comparison with the molecular-weight labels ➫ Can be analyzed by sequencing of the amplified DNA, after extraction from agarose ➫ The amplified fragment does indeed correspond to the expected 427 bp fragment, as can be seen in lines 2, 3, and 5. Line 2 = PCR on the positive-control DNA Line 3 = PCR specific to the DNA being studied Line 4 = Negative PCR: No band is visible Line 5 = Nonspecific PCR: The parasite bands are due to illegitimate hybridizations between primers and DNA Figure 5.8 Analysis of amplification products on agarose gel. 101

Polymerase Chain Reaction (PCR) When the amplified product has been obtained, liquid-phase PCR is also an excellent way of optimizing the amplification conditions which often reduces to looking for the optimal primer hybridization temperature. For example, raising the hybridization temperature by a few degrees may be enough to eliminate the parasite bands. 50

52

54

56

60°C

58

➫ This “ideal” temperature is the starting point for the adjustment of the in situ PCR.

➫ The temperature should be that which gives the best signal without parasite bands (in this case, 56°C).

427 bp

Figure 5.9 Optimization of the primer hybridization temperature. If, in spite of this rise in temperature, there are still parasite bands, it will be necessary to: • Reduce the number of cycles • Lower the concentration of the primers If these different modifications bring about no improvement, other primers will have to be found before in situ PCR can be carried out.

5.3.2.7

➫ From 30 to 25 or 20 cycles ➫ Which, in excess, can hybridize in a nonspecific way ➫ In in situ PCR, if one or both primers hybridize in a nonspecific way, it is impossible to tell false positives from valid results, and the data are impossible to interpret.

Labeled primers

The use of labeled primers in a direct PCR protocol is considered as the least specific method, and is not recommended. It is, however, potentially useful, notably with fluorescent labels.

35

➫ See Section 9.4. ➫ See Chapter 11, Figure 11.1. ➫ Ease of use and rapidity ➫ The labeling of the primer must be carried out in the 5′′ position, or through the incorporation of labeled nucleotides during oligonucleotide synthesis. The labeling system depends on the particular label used. Labeling in the 3′ position leads to the hybridization of this end of the target sequence. The positioning of the enzyme reduces the efficiency of the extension, and thus the amplification.

33

5.3.2.7.1 RADIOACTIVE LABELS ( S OR P) The labeling uses the enzymatic activity of the polynucleotide kinase to insert a radioactive nucleotide at the 5′ position of the primer. This nucleotide is itself labeled on its phosphate group at the γ position. The labeling process thus consists of phosphorylating the 5′ end by adding a phosphate.

102

➫ And thus more specific hybridization

➫ γ[ S]-dATP (or -dCTP) and γ[ P]-dATP (or -dCTP) are available from most suppliers of molecular biology products. 35

33

5.3

Tools

➫ S or P labeling of the phosphate group in the γ position 35

33

➫ The addition reaction is carried out by polynucleotide kinase (an enzyme extracted from calf thymus).

Figure 5.10 Labeling by 5′ extension.

Besides the lack of specificity of the direct method, serious contamination problems have been mentioned in relation to radioactivity, and 35 in particular with S. Those who insist on using radioactivity should opt 33 for P. 5.3.2.7.2

➫ Probably due to the emission, at high temperatures, of radioactive H2S ➫ Whose half-life is relatively short (25 days)

ADVANTAGES/DISADVANTAGES

❑ Advantage • Sensitivity ❑ Disadvantages • The cost, along with the specific problems inherent in the manipulation of radioactive substances 33 • Low cell resolution; with P, it is around 15 to 20 µm

➫ There is a need for radioprotection. ➫ This is a major disadvantage, as the aim of in situ PCR is to identify the cells that express the gene of interest within a given cell population.

• Low specificity due to the use of the direct method

103

Polymerase Chain Reaction (PCR) 5.3.2.7.3 NONRADIOACTIVE LABELS Antigenic molecules such as biotin, digoxigenin, alkaline phosphatase, and the fluorescent molecules are either: s

• Chemically coupled to the modified 5′ end of a synthetic oligonucleotide, in which case the addition of an aliphatic amine group to the 5′ end makes possible the conjugation of different molecules, or • Incorporated during the synthesis of the oligonucleotide. Several labeled nucleotides can thus be incorporated, and this increases the efficiency of the labeling. 5.3.2.7.4

➫ This amine modification of the 5′ end of the synthetic oligonucleotide is available from a number of suppliers. ➫ Several labels are also available at the 5′ end, e.g., biotinylation, fluorescein, rhodamine, alkaline phosphatase, peroxidase. ➫ This method is used mostly for fluorescent labels. ➫ It reduces the efficiency of the hybridization process.

ADVANTAGES/DISADVANTAGES

❑ Advantages • Easy to use • Rapid • Two PCRs can be carried out simultaneously, using primers labeled with fluorochromes of different colors.

➫ Specific equipment is not required. ➫ This option, which is used mostly with cell suspensions, makes it possible to visualize two sequences of different genes in the same cell or different cells.

❑ Disadvantages • This method does not give the same degree of specificity as the direct method. • The observation of fluorescence requires a suitable microscope. • It is impossible to conserve samples processed by fluorescence.

5.3.3

Pfu DNA polymerase, which has a different origin and has appeared on the market more recently, presents some advantages.

104

➫ Confocal microscopy provides a satisfactory degree of precision.

Enzymes

The most commonly used enzyme is Taq DNA polymerase, which can be obtained from any supplier of molecular biology products, modified to a greater or lesser extent to maximize its thermal stability, ease of use, and effectiveness. 

➫ See Section 9.2.

➫ It is derived from a thermophilic bacterium, Thermus aquaticus, which lives in thermal springs at 70°C. ➫ All the different suppliers now offer complete ranges of enzymes, corresponding to the diversity of users’ needs. ➫ This is derived from Pyrococcus furiosis, which flourishes at 100°C in geothermal marine sediments. ➫ It is very stable. ➫ Its level of DNA polymerase activity is very low at temperatures <50°C, which avoids hot starts.

5.3

Tools

The aim, here, is not to present an exhaustive, comparative list of all currently available enzymes, but to set out their most significant––and most useful––properties. 5.3.3.1

Taq DNA polymerase

❶ Origin This thermostable DNA polymerase is most often recombinant. It is obtained from the recombinant bacterial strain E. coli, which expresses the YT1 gene of Thermus aquaticus. Its molecular weight varies between 85 and 94 kDa. ❷ Presentation The quantity corresponds to a level of activity of 250 to 1000 U. Its concentration is generally 5 U/µl. Composition of the conservation buffer is the following: 20 mM Tris–HCl, pH 8 (25°C); 100 mM KCl; 0.1 mM EDTA; 1 mM DTT; 50% glycerol; 0.5% Tween 20; 0.5% Igepal. A blue or red stain is sometimes added to this   buffer (e.g., Eurobluetaq or Red Taq ).

Most enzymes are delivered at room temperature, which is possible due to their high degree of thermal stability. The enzyme is supplied with its corresponding reaction buffer; the supplier’s guarantee depends on its use. The MgCl2 solution, which is a cofactor of the enzyme, is supplied separately. In this way it can be used at a range of concentrations to optimize its effectiveness. ❸ Conservation For the enzyme to remain at its maximal level of activity, it should be stored at −20°C. Some manufacturers guarantee 2 years of stability in such conditions. ❹ Principle of action Taq DNA polymerase catalyzes the polymerization of nucleotides into double-strand DNA in the 5′ → 3′ direction in the presence of Mg. Some of them display 5′ → 3′ exonuclease activity. Some enzymes make possible the extension of large fragments, and the user’s choice will take into account, among other things, the stated effectiveness.

➫ Cloning the enzyme allows a high degree of reproducibility between one batch and another. ➫ The weight depends on how it is produced. ➫ A unit is defined as the quantity of enzyme needed to transform 10 nmol dNTP into insoluble DNA in 30 min at 74°C in an acid medium. ➫ The 50% glycerol prevents freezing at −20°C. ➫ This eliminates the risk of forgetting about the enzyme. ➫ This is a way of checking the homogenization of the enzyme in the reaction buffer. ➫ To increase the thermal stability of their enzymes, some manufacturers carry out aminoacid substitutions in several places at the N terminal end of the protein. ➫ Concentration: 10X ➫ Concentration: 50 mM

➫ To avoid successive reheatings, an isothermal refrigerated box should be used.

➫ The supplier should guarantee that the enzyme has no contaminating endonuclease activity and should state whether or not it has any exonuclease activity. ➫ Some enzymes can amplify fragments of 100 bp to 20 kb, but in most cases the enzyme should be suited to the desired extension. 105

Polymerase Chain Reaction (PCR) For in situ PCR and RT-PCR, the most important properties for an enzyme to possess are: • Processivity • Fidelity

• Thermal stability ❺ Utilization • Reaction buffer Reaction buffers are adapted and optimized so that the enzyme will be optimally effective. The components of a given buffer tend to be more or less the same from one supplier to another, but their concentrations may vary considerably. • Concentration It must be optimized; 0.05 U/µl is a good starting point. • Cofactor The MgCl2 concentration must be empirically determined by making a range of increasing concentrations. 2+ Mg influences the enzyme activity and the stabilization of the double strand, and raises the Tm. It also forms soluble complexes with dNTPs, which are recognized and utilized by the enzyme.

➫ For in situ PCR, the size of the fragment to be amplified should be >300 bp, but never >1 kb. ➫ Taq DNA polymerases incorporate, on average, 500 bp in 15 to 20 s. ➫ The general error rate for a Taq DNA poly−4 merase is of the order of 10 (i.e., one erroneous nucleotide per 10,000 incorporations). ➫ At 95°C, the half-life varies between 1 and 3 h, depending on the enzyme. ➫ For example: 100 mM Tris-HCl, pH 8.8; 500 mM KCl; 1% Triton X100

➫ 2.5 to 5 U/50 µl reaction buffer ➫ Or, with some suppliers, MgSO4 ➫ 0.5 to 4 mM ➫ MgCl2 is complexed mole by mole with EDTA. –– An excess of MgCl2 reduces the fidelity of the enzyme. –– A lack of MgCl2 reduces the reaction yield.

Final MgCl2 Concentration

106

1.5 mM

2 mM

2.5 mM

3 mM

3.5 mM

4 mM

Final Concentration

× Buffer 10×

10 µl

10 µl

10 µl

10 µl

10 µl

10 µl

1X

MgCl2

3 µl

4 µl

5 µl

6 µl

7 µl

8 µl

1.5–4 mM

dNTP

10 µl

10 µl

10 µl

10 µl

10 µl

10 µl

250 µM

Enzyme

1 µl

1 µl

1 µl

1 µl

1 µl

1 µl

5U

Sense primer

5 µl

5 µl

5 µl

5 µl

5 µl

5 µl

0.5 µM

Anti-sense primer

5 µl

5 µl

5 µl

5 µl

5 µl

5 µl

0.5 µM

H2O

66 µl

65 µl

64 µl

63 µl

62 µl

61 µl

Final volume

100 µl

100 µl

100 µl

100 µl

100 µl

100 µl

5.3 • Temperature Taq DNA polymerase attached to the 3′ end of a primer synthesizes a strand complementary to each of the original strands at an optimal temperature of 72 to 74°C (depending on the supplier).

❻ Inhibition of DNA polymerase activity at low temperatures • By the addition of an antibody: Taq DNA polymerase is placed in the presence of an antibody, which inhibits its action up to the first denaturation cycle, where the temperature of >90°C, dissociates the enzyme/ antibody complex. When the antibody is denatured, Taq DNA polymerase activity is restored. • By the use of paraffin micropellets containing MgCl2: During the first denaturation cycle, 2+ the paraffin melts, thus liberating the Mg needed for the enzyme to work properly. 5.3.3.2

Tools

➫ The activity of the polymerase is optimal at around 74°C, but it begins to appear at much lower temperatures, thus causing primer mismatching, which leads to undesired extensions and, in the end, artifactural amplification products. It was to avoid this disadvantage that the hot start technique was developed, and some manufacturers have produced quite ingenious systems. ➫ This system limits the risk of nonspecific amplifications. ➫ The use of this enzyme avoids hot starts.

➫ With this system, hot starts do not occur.

Other enzymes

Here, only the distinctive (and useful) properties of other enzymes are presented. 5.3.3.2.1



PFU

AND



TGO DNA POLYMERASE

❶ Origin Whether native or cloned, these thermally stable DNA polymerases are derived from Pyrococcus furiosis, which lives at 100°C in geothermal marine environments. ❷ Characteristics • Thermal stability:  Pfu DNA polymerase has a half-life of 18 to 25 h at 95°C.

• Reduced polymerase activity below 50°C, which avoids hot starts.

➫ It is their origin that gives them their high level of thermal stability.

➫ This extreme thermal stability means that denaturation temperatures as high as 98°C are possible, with target sequences that are rich in GC. ➫ This property reduces the number of extensions resulting from nonspecific primer mismatching, which frequently occurs with Taq DNA polymerase, whose activity is high at temperatures below 50°C (i.e., the average primer hybridization temperature).

107

Polymerase Chain Reaction (PCR) ➫ The activity of the two recombinant enzymes is compared, for equal concentrations and for temperatures of 15 to 100°C. ➫ Taq DNA polymerase activity increases from 17 to 70% between 35 and 50°C, whereas  activity of Pfu DNA polymerase increases only from 2 to 8% between 35 and 50°C.

Taq DNA Polymerase Pfu DNA Polymerase Polymerase activity percentage

100 80 60 40 20



0 15

35

50 60 70 80 Temperatures

95

Figure 5.11 Comparison of Taq and Pfu DNA polymerase activity as a function of temperature.

(Data provided by Stratagene.) 5.3.3.2.2



EXTRAPOL DNA POLYMERASE

❶ Origin This enzyme is obtained from the recombinant bacterial strain E. coli, which expresses the Thermus brockianus gene. ❷ Characteristics • Thermal stability Extrapol has a half-life of 3 h at 96°C. • Fidelity Its error rate is only half that of a classical Taq DNA polymerase. 5.3.3.2.3

Tth DNA POLYMERASE

❶ Origin This is a recombinant enzyme that is cloned from the bacterial strain Thermus thermophilus. ❷ Characteristics • It catalyzes the polymerization of nucleotides in double-strand DNA, in the presence of MgCl2. • It also polymerizes DNA, using an RNA template in the presence of MnCl2.

108

➫ For greater thermal stability

➫ See Section 4.3.3.3.

➫ DNA polymerase activity ➫ Reverse transcription activity

5.4 5.3.3.3

Equipment/Reagents/Solutions

Criteria of choice

Enzyme

Classical Taq DNA Polymerase

Origin and method of production





Extrapol

Pfu or Tgo DNA Polymerase

Tth DNA Polymerase

Thermus aquaticus (cloning)

Thermus brokianus (extracted or cloned)

Pyrococcus furiosus (extracted or cloned)

Thermus thermophilus (cloned)

Concentration

5 U/µl

5 U/µl

2.5–5 U/µl

5 U/µl

Transport temperature

Room temperature

Room temperature

Room temperature

Room temperature

Storage temperature

–20°C

–20°C

–20°C

–20°C

Maximum size of the fragment to be amplified

1.8 kb

6–25 kb

100 bp–40 kb

12 kb

Thermal stability (halflife at 95°C)

1h

3h

18–25 h

1h

Optimal operating temperature

72°C

72°C

72°C

72°C

2+



2+

Mg

2+

2+

Cofactor

Mg

Mg

Mg

Reaction buffer pH

8.8

8.8

8.75

8.8

5′ → 3′ exonuclease activity

Yes

Yes

Yes

Yes

Processivity

15–20 s/500 bp

40–45 s/kb

40–45 s/kb

15–20 s/500 bp

Fidelity (error rate)

Classical −4 (10 )

High fidelity −5 (3.6 × 10 )

High fidelity −7 (4.9 × 10 )

Classical −4 (10 )

Stability

24 months

24 months

24 months

24 months

5.4 EQUIPMENT/REAGENTS/SOLUTIONS 5.4.1 Thermocyclers

➫ See Section 4.4.1.

It is ease of programming and reliability that will determine the choice of apparatus.

➫ All the various types of apparatus give similar results, essentially differing only in terms of the number of slides they hold. 109

Polymerase Chain Reaction (PCR) The speed with which the temperatures of the different programmed stages are attained and the homogeneity of the temperature within the apparatus are also criteria for choice.

5.4.2 Sealing Equipment For the reverse transcription step, the section must be covered with a sealed incubation chamber that will withstand the high-temperature PCR cycles. 5.4.2.1

• Use the Easyseal system.

➫ The least sophisticated method; also the least reliable ➫ Specifically designed for the Perkin-Elmer Applied Biosystems thermocycler (see Section 4.4.2.1) ➫ Suitable for all other types of thermocycler (see Section 4.4.2.2)

Sealing apparatus

Apparatus is designed to carry out hot starts very easily. The heating block system over which the slide moves maintains the section and the reaction medium at a temperature of 95°C, thus reducing the risk of nonspecific primer hybridization.

4 3

2 6

7

5

8

110

➫ 25 cycles, each lasting, on average, 4 min, at temperatures of 55–95°C

The different systems

Place cover slips on the sections (which have been covered with the reaction medium) and seal them with a silicone of the rubber cement type. • Use the “cover disk/cover clip” system.

5.4.2.2

➫ See Section 4.4.2.

1

➫ See Figure 4.13 and Section 4.5.2.1.1.

① Runners in which the slide moves as the cover disk/cover slip system is put in place ② Heating block at a constant temperature of 95°°C (denaturation temperature) ③, ④ Cover disk/cover slip system held in place on the magnetic plate ⑤ Red light: Heating-block indicator ⑥ Green light: Indicates that the temperature has attained 70°°C; with this system, it is easy to carry out a hot start before the amplification step ⑦ Blocking system ⑧ Compression wheel Figure 5.12 “Assembly tool” sealing apparatus (PE Applied Biosystems). Open position.

5.4

Equipment/Reagents/Solutions

5.4.3 Reagents/Solutions • Sense and anti-sense primers

• Deoxynucleotide triphosphates (dNTP)

• Enzyme

• A reaction buffer that is specific to the enzyme without MgCl2, or at a minimum concentration of 1.5 mM It is essentially made up of: — Tris-HCl — 500 mM KCl, to which may be added:

— A detergent — EDTA — DTT

• MgCl2 solution This is supplied separately in tubes, and its final concentration in the reaction buffer needs to be optimized. • DMSO (dimethylsulfoxide) If the sequences are rich in GC (>80%), secondary structures may form, which will reduce the PCR yield. Adding DMSO prevents the formation of such structures. • Formamide

➫ See Section 5.3.2. ➫ Starting with a 100 mM solution, prepare a 10 µM storage solution. ➫ Store at –20°C. ➫ This is available separately, or in the form of a “mixture” at a concentration of 100 mM. ➫ Starting with a 100 mM solution, prepare a 10 µM storage solution. ➫ Store at –20°C. ➫ This is generally delivered at room temperature due to its high level of thermal stability. ➫ Store at −20°C. Each enzyme has its own particular characteristics, and it is important to follow the manufacturer’s instructions. ➫ Its composition is optimized by the manufacturer. It is generally supplied in 10X form. ➫ Store at −20°C. ➫ This is at different concentrations, and a pH of 8.3 to 9, according to the enzyme. ➫ The pH of the reaction medium has a decisive influence on PCR efficiency. The pH that gives optimum fidelity is 8.3; that which gives optimum sensitivity is >9. The usual compromise is somewhere around 8.8, at 25°C. ➫ Ionic detergents (Tween 20, Triton ×100, Nonidet P40) can be used at a concentration of 0.05% to stabilize amplification enzymes. At higher concentrations they inhibit DNA polymerase activity. If the experiment requires an excess of detergent, it is advisable to increase the enzyme concentration. ➫ Storage is at −20°C. ➫ This is supplied at a concentration of 50 mM. ➫ See table in Section 5.3.3.1.

➫ A concentration of 5 to 10% of the final reaction volume. ➫ A concentration higher than 10% can totally inhibit enzyme activity.

111

Polymerase Chain Reaction (PCR) Some primer sequences may necessitate a high Tm, i.e., a temperature close to the optimal activity temperature of the enzyme (72°C). This will affect the amplification. Adding formamide lowers the reaction temperature. • Sterile water

➫ Tm: melting temperature. ➫ At a concentration that has to be determined empirically. As with DMSO, a high concentration will affect the activity of the enzyme. ➫ The quality of the water is very important: DEPC water (see Appendix B1.2); 2 ml ampoules of sterile water are very practical.

5.5 PROTOCOL The in situ PCR step can be carried out by different methods, using: • Tissue sections, either frozen or embedded in paraffin • Cells, either cytocentrifuged or cultured on slides • Cells in suspension, whether from cultures, biological fluids, or even the enzymatic dissociation of tissue

➫ See Section 2.1.

This amplification step is carried out either: • Directly, after the pretreatment of the different samples, with the amplification of a DNA target sequence, or • After the target mRNA sequence has been transformed by reverse transcription into DNA

5.5.1

Reaction Mixture

5.5.1.1

Direct PCR

There are two ways of carrying out direct PCR:

➫ See Chapter 3. ➫ See Chapter 4.

➫ Gloves must be worn. ➫ RNase-free conditions are very important.

• The incorporation of a labeled dNTP, or • The use of labeled primers ❶ Reaction medium using a labeled dNTP In a microtube placed in ice, prepare the following mixture: • PCR buffer (10X) • Labeled dATP or dUTP (0.4 mM) • Unlabeled dATP or dCTP (10 mM) • A dNTP other than the labeled dNTP (10 mM) 112

1X ≈100 µ M ≈100 µ M ≈200 µ M

➫ According to the manufacturer ➫ Generally biotin-14-dATP or digoxigenin11-dUTP ➫ Complementary to labeled dATP or dUTP ➫ The three other deoxynucleotides added at the same final concentration

5.5 • Sense primer (10 µM)

0.3–0.5 µ M

• Anti-sense primer (10 µM) • MgCl2 (50 mM) • Taq DNA polymerase (5 U/µl)

0.3–0.5 µ M 1.5–4 mM µl 1–2.5 U/µ

Protocol

➫ The same concentrations for the sense and anti-sense primers ➫ Necessary to optimize the final concentration ➫ According to the enzyme ➫ Added only after 5 min of incubation at 82°C, for a hot start

• Sterile water

To a total volume of 100 µl ❷ Reaction medium using labeled primers In a microtube placed in ice, prepare the following mixture: • PCR buffer (10X) • dNTP (10 mM )

1X ≈200 µ M

• Labeled sense primer (10 µM)

≈1 µ M

• Labeled anti-sense primer (10 µM)

≈1 µ M

• MgCl2 (50 mM) • Taq DNA polymerase (5 U/µl) • Sterile water

5.5.1.2

1.5–4 mM µl 0.1–0.3 U/µ

➫ According to the manufacturer ➫ The four deoxynucleotides added at the same final concentration ➫ Either biotinylated, fluorescent, or digoxigenin labeled ➫ Possible also to use a radioactive label (e.g., 35 33 S or P) ➫ Either biotinylated, fluorescent, or digoxigenin labeled ➫ Possible also to use a radioactive label (e.g., 35 33 S or P) ➫ Necessary to optimize the final concentration ➫ According to the enzyme ➫ Added only after 5 min of incubation at 82°C, for a hot start

To a total volume of 100 µl

Indirect PCR/RT-PCR

In a microtube placed in ice, prepare the following mixture: • PCR buffer (10X) • dNTP (10 mM)

• • • •

Sense primer (10 µM) Anti-sense primer (10 µM) MgCl2 (50 mM) Taq DNA polymerase (5 U/µl)

• Sterile water

1X ≈200 µ M ≈1 µ M ≈1 µ M 1.5–4 mM µl 0.1–0.3 U/µ

➫ According to the manufacturer ➫ The four deoxynucleotides added at the same final concentration ➫ Separately or in a “mixture” ➫ Necessary to optimize the final concentration ➫ According to the enzyme ➫ Added only after 5 min of incubation at 82°C, for a hot start

To a total volume of 100 µl 113

Polymerase Chain Reaction (PCR)

5.5.2 The Hot Start This step is designed to avoid mismatched extension reactions. The fact is that classical Taq DNA polymerase displays a relatively high level of DNA polymerase activity at >30°C. 5.5.2.1

Procedure

a. Prepare the reaction mixture without Taq DNA polymerase. b. Incubate the reaction mixture. 5 min 82°°C c. Add Taq DNA polymerase to the reaction mixture. >70°°C d. Place the dry, pretreated slides on the heating plate of the sealing apparatus, or a heating block. e. Immediately place 30 µl of the reaction mixture on the denatured sections. f. Cover the sections with Ampli cover disks and cover clips (Perkin-Elmer Applied Biosystems), or Easyfilm (Hybaid). g. Place the slides in the preprogrammed thermocycler, and begin with the denaturation step.

5.5.2.2

➫ But with the necessary quantity of sterile water, to a total volume of 100 µl. ➫ This temperature can be higher, depending on the thermal stability of the enzyme. ➫ This temperature limits the number of nonspecific hybridizations. ➫ Double-strand DNA is not denatured, and a temperature of >90°C is necessary. ➫ See Section 4.4.2. ➫ Or simply use sterile cover slips, carefully sealed. A considerable amount of evaporation takes place during the high-temperature cycles. ➫ It is a good idea to leave the apparatus at 82°C while the reaction mixture is placed on the sections and the slides are sealed. This means that the first cycle can start as soon as the slides are ready.

Avoiding hot starts

A hot start is a demanding technique, but various manufacturers have developed procedures and new enzymes that allow it to be avoided, while achieving a higher quality of amplification. ❶ Blockage of Taq DNA polymerase activity by an anti-Taq DNA polymerase antibody ❷ MgCl2 embedded in paraffin micropellets, as a cofactor of the enzyme





❸ New enzymes: Pfu and Tgo DNA polymerase, whose activity is very low at <50°C

114

➫ It is at lower temperatures (37 to 40°C) that nonspecific hybridizations occur spontaneously.

➫ It is during the denaturation step in the first PCR cycle that the antigen/antibody complex breaks down, thus restoring the enzymatic activity of the Taq DNA polymerase. ➫ The paraffin melts at around 55°C, liberating the MgCl2. The enzyme is thus active only above this temperature, which is close to the specific hybridization temperature of most primers. ➫ See Section 5.3.3.2. ➫ Some of these enzymes are also coupled to an antibody to maximize the chances of obtaining satisfactory results.

5.5

Protocol

5.5.3 The Amplification Cycles The PCR reaction takes place in a three-phase cycle. ❶ Denaturation phase At a temperature of 90 to 95°C, the helical double strand that makes up the DNA template linearizes, and the two strands separate out. The success of this step will determine that of the entire experiment. • Temperature and duration: The degree of denaturation depends 92–97°°C <1 min on the temperature, and the length of time for which it is maintained. ❷ Hybridization phase At a well-defined temperature (Tm), the pair of primers hybridizes in a specific way, each on one of the two DNA strands, thereby specifying the part of the target sequence that will be amplified. • Temperature and duration: . 50–70°°C The hybridization temperature 30–60 s depends on the nature, length, and concentration of the primers. It is 5 to 10°C below the Tm. ❸ Extension phase DNA polymerase become attached to the 3′ end of each primer, and synthesize the complementary strand to each of the two DNA strands. • Temperature and duration: At 72°C, DNA polymerases can 72°°C incorporate 25 to 50 nucleotides/s, which means that the duration of the 1 min extension step depends on the length of the fragment to be amplified.

➫ If the denaturation is only partial, a return to the double-strand form will prevent the hybridization of the primers from taking place, and will lead to a false-negative result. ➫ 92°C for 30–60 s ➫ 97°C for 15–30 s ➫ See Section 5.3.2.

➫ For a given PCR, the Tm of the two primers should be as close together as possible. ➫ It should be noted that the closer the hybridization temperature is to the optimal working temperature of the in vitro amplification enzymes, the greater the efficiency of the PCR.

➫ The optimal working temperature for DNA polymerases ➫ 15 to 30 s if the fragment to be amplified is <500 bp, 45 to 60 s if it is >1 kb

5.5.4 Number of Cycles The amount of amplification––in other words the number of copies of a given target sequence––is directly proportional to the number of cycles. In situ amplification protocols generally comprise 20 to 30 cycles. The precise number depends on:

➫ Number of copies = 2 , where n is the number of cycles.

• The quantity of target DNA that is present in the tissue

➫ For target sequences of which there are few copies, the number of cycles can be increased, although this of course entails an increased risk of nonspecific amplification.

n

➫ The average number is 25, but the optimal number can only be determined empirically.

115

Polymerase Chain Reaction (PCR) ➫ Beyond 20 cycles, the morphology of the tissue is often altered, which means that the risk of diffusion increases, with interpretational difficulties as a consequence.

• The degree of fragility of the tissue

5.5.5 Programming the Thermocycler The three steps that make up a PCR cycle take place at precise temperatures: 95°°C 50–70°°C

• Denaturation • Hybridization

72°°C

• Extension

The programming of the thermocycler makes it possible to go from one temperature to another automatically, to maintain given temperatures for given lengths of time, and to repeat a cycle a given number of times. • Denaturation • Hybridization • Extension

30–60 s 60–90 s 60–90 s

➫ See Chapter 1 and Section 5.5.3. ➫ See Section 5.5.3. ➫ According to the primers used (see Section 5.5.3). ➫ See Section 5.5.3. ➫ It is the rapidity, precision, and reproducibility of the thermocycler that determine the yield and quality of the amplification step.

➫ According to the temperature ➫ According to the temperature ➫ According to the length of the amplified product

Temperatures (°C)

A time increment can be factored in for higher numbers of cycles. 100 90 80 70 60 50 40 30 20 10 0

① Denaturation: 30 s at 94°C

1

② Hybridization: 60 s at 55°C

3 2

③ Extension: 60 s at 72°C

1st cycle

2nd cycle

Figure 5.13 PCR programming diagram.

Time (s)

The first cycle begins when the temperature reaches 94°C, after a phase during which the apparatus was maintained at a temperature of >80°C so as to carry out a successful hot start. At the end of the last cycle it is necessary to program: • A final extension phase

116

72°°C 5 min

➫ The time needed to prepare the slides and load them into the apparatus

5.5 • A temperature at which the slides can be held for processing

4°°C

Protocol

➫ Experience shows that after spending a certain time at 4°C, sealing systems are difficult to remove, and that there is a high risk of spoiling the section. The temperature should therefore be raised (to 40 to 50°C) before opening the apparatus.

5.5.6 The Particular Case of Cells in Suspension The cells are either pretreated or washed after the reverse transcription phase. In both cases, they are in suspension in a phosphate buffer. a. Centrifuge, then remove the 2 min supernatant. 1500 g b. Add the reaction mixture to the cell pellet, and homogenize by delicate pipetting. c. Incubate the mixture. 5 min >80°°C d. Add the enzyme. e. Place in a thermocycler programmed for 20 to 25 amplification cycles, having checked that the tubes are firmly closed. f. Wait a final extension phase. 5 min 72°°C g. Stop the reaction. 10 s 30°°C

➫ See Section 2.2. ➫ See Section 4.5.2.2.

➫ For a hot start, this reaction mixture should not contain any DNA polymerase.

➫ Final concentration: 0.1 to 0.3 U/µl

➫ The microtubes can be kept waiting for some time after the thermocycler is programmed at 4°C.

5.5.7 Washing 5.5.7.1

Washing and Postfixation of sections or cells on slides

a. Remove the cover disk/cover slip, the Easyseal system, or the sealed coverslip. b. Wash in 0.1 M sterile phosphate buffer. c. Postfix:

• 4% paraformaldehyde, or • 70° cold alcohol

5 min

10–15 min 10 min −20°°C

➫ This is a delicate step, during which the tissue section or the cells risk damage via a suction effect. Detach as gently as possible. ➫ This step is carried out in a tray. ➫ This postfixation step is necessary to the fixation of the amplified products. It also stabilizes tissue structures. ➫ For tissue sections, see Appendix B4.3.2. ➫ Use for cell cultures on slides, or smears.

d. Rinse: • 0.1 M phosphate buffer • 9‰ NaCl

5 min 2 min

➫ See Appendix B3.4.1.

117

Polymerase Chain Reaction (PCR) e. Dehydrate in alcohol baths of 2 min increasing concentration: 95°, per bath 100°. f. Allow the slides to dry under a 30–60 min ventilated hood. ➲ Following steps • Direct observation of the amplified product

• Hybridization of the amplified product with labeled probes • Antigenic detection of the amplified product

5.5.7.2

➫ After direct PCR with primers or fluorescent dNTP ➫ The amount of amplification product obtained is rarely enough to permit direct visualization. Antigenic detection with an antifluorescein will then need to be considered. ➫ After indirect PCR ➫ See Chapter 6. ➫ After direct PCR ➫ See Chapter 7.

Washing cells in suspension

a. Centrifuge.

2 min 1500 g b. Remove all the supernatant, and resuspend in around 200 µl PBS. c. Centrifuge. 2 min 1500 g d. Remove the supernatant, and resuspend in 1000 µl PBS. ➲ Following steps • Observation after spreading the suspension on slide, or by flow cytometry • Hybridization in the case of indirect PCR with unlabeled primers • Antigenic detection if the label is biotin or digoxigenin

118

➫ See Appendix B3.4.3.

➫ See Appendix B3.4.3. ➫ The amplification product is directly visible if the amplification has been carried out in the presence of dNTP or fluorescent primers. ➫ Hybridization can be carried out either in tubes or after spreading the suspension on slides (see Chapter 6). ➫ After spreading the suspension on slide (see Chapter 7).

Chapter 6 Hybridization

Contents

CONTENTS 6.1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

123

6.2

Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . .

124

6.3

Tools: The Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

125

6.3.1 6.3.2

125 125 125 125 126 126 126 127 127 128 128 129 129 129 130

6.3.3

Types of Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2.1 Complementarity . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2.2 Anti-sense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2.3 Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2.3.1 Oligonucleotide Probes. . . . . . . . . . . . 6.3.2.3.2 cDNA Probes . . . . . . . . . . . . . . . . . . . Labeling the Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.1 Antigenic Labels . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.2 Radioactive Labels . . . . . . . . . . . . . . . . . . . . . . . . 35 S............................ 6.3.3.2.1 33 6.3.3.2.2 P............................ 6.3.3.3 Labeling by PCR. . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 6.3.3.3.2 Protocol. . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.3.3 Reaction Mixture for Antigenic Labeling . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.3.4 PCR Protocol . . . . . . . . . . . . . . . . . . . 6.3.3.4 Labeling by 3′ Extension . . . . . . . . . . . . . . . . . . . 6.3.3.4.1 Overview 6.3.3.4.2 Equipment/Reagents/Solutions. . . . . . 6.3.3.4.3 Protocol for Radioactive Probes . . . . . 6.3.3.4.4 Protocol for Antigenic Probes. . . . . . . 6.3.3.5 Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.5.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 6.3.3.5.2 Equipment/Reagents/Solutions . . . . . 6.3.3.4.3 Protocol. . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.6 Controls/Storage/Utilization . . . . . . . . . . . . . . . . 6.3.3.6.1 Checking the Labeling . . . . . . . . . . . . 6.3.3.6.2 Storage . . . . . . . . . . . . . . . . . . . . . . . . 6.3.3.6.3 Utilization . . . . . . . . . . . . . . . . . . . . . .

131 131 132 132 133 134 135 135 135 136 136 137 137 137 137

121

Hybridization 6.4

Hybridization Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

138

6.4.1

Hybridization Temperature . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.1.1 Melting Temperature (Tm) . . . . . . . . . . . . . . . . . . 6.4.1.2 The Difference between Tm and the Hybridization Temperature . . . . . . . . . . . . . . . . . . + Na Ion Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hybridization Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Nature of the Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . Probe Length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

138 138

Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

140

6.5.1

Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.1.1 Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.1.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Reaction Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

140 140 141 141 142

Posthybridization Treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

143

6.6.1 6.6.2

Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6.2.1 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 6.6.2.2 Na Ion Concentration . . . . . . . . . . . . . . . . . . . . . 6.6.2.3 Washing Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

143 143 144 144 144 144

Before Revelation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

145

6.7.1

145 145 145 145

6.4.2 6.4.3 6.4.4 6.4.5 6.4.6 6.5

6.5.2 6.5.3 6.6

6.6.3 6.7

6.7.2

122

Radioactive Hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.1.1 Dehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.1.2 Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antigenic Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

138 140 140 140 140 140

6.1

The hybridization step in the in situ PCR/ RT-PCR protocol is necessary only in the indirect method, where the amplified product is present in the cells in the form of doublestranded DNA. Its purpose is the visualization of this amplified product by a specific detection method, namely, in situ hybridization.

Overview

➫ In the direct method, the label is incorporated during the synthesis of the amplified product. ➫ This method can be used without amplification to visualize DNA or RNA in situ.

6.1 OVERVIEW Two labeled probes, each of which is complementary to one of the two strands of neosynthesized DNA, hybridize to form labeled hybrids. The hybridization reaction does not require any input of energy.

It is due to the formation of hydrogen bonds between the different bases that make up the sequences of one of the strands of the amplified product and the probe that is complementary and anti-sense to it. The fact that the probe is labeled means that the complex or hybrid is also labeled, and this allows it to be detected.

➫ Hybridization is the basis of the RT and PCR steps (see Chapters 4 and 5). ➫ This is always the case for molecular hybridization. ➫ The opposite of hybridization is denaturation, which occurs at the melting point, and consists of using heat to separate the two strands of the hybrid. ➫ There are two bonds between A and T, and three between G and C.

➫ For the labeling of the probe, see Section 6.3.3. ➫ For the detection process, see Chapter 7. 123

Hybridization

3'

3'

5'

5'

① Denaturation of the amplified DNA

1 5'

3'

3'

5'

2

② Hybridization of the two labeled probes on the two strands of DNA 3'

5' 3' 3'

5'

5'

3'

5'

3

③ Labeled hybrids

Figure 6.1 The hybridization step.

6.2 DIAGRAM OF THE DIFFERENT STEPS Fixation of amplified product

↑↓

Labeling the probes

Denaturation

Hybridization

Washes

Labeled hybrids

124

6.3

Tools: The Probes

6.3 TOOLS: THE PROBES A probe is a nucleic acid whose sequence is determined by: • Complementarity • Anti-sense • Specificity Two probes, anti-sense and sense, are necessary, given that the amplified product is double stranded.

➫ Of each strand of the amplified product

➫ Hybridization can, in fact, be carried out with a single probe, but in this case the detection will be reduced by half.

6.3.1 Types of Probes The nature of the probes is not a limiting factor as such. However, simplicity should be favored, which is the reason the most widely used probes are: • Synthetic oligonucleotides • cDNA sequences

• cRNA

➫ These are easily obtained and labeled, and give efficient hybridization. ➫ These consist of double strands of DNA, each of which can hybridize with one of the two strands of the amplified product. They are most often produced by a PCR in the liquid phase. A nested PCR using the amplified product can give smaller-sized probes. ➫ These are used only exceptionally.

6.3.2 Characteristics The characteristics of the probes must satisfy the following criteria: • Complementarity • Anti-sense • Specificity 6.3.2.1

Complementarity

The sequence of the two strands of neosynthesized DNA is known, and the structure of the complementary sequences is identical to that of primers. 6.3.2.2

➫ For primers, specificity is not crucial [poly (A)], but for probes it must be carefully checked.

➫ See Section 4.3.1.

Anti-sense

For hybridization to take place, the sequence of the probe must be anti-sense to the nucleotide sequence to which it is complementary.

➫ See Section 4.3.1.

125

Hybridization 6.3.2.3

Specificity

It is the specificity of the probes that defines the specificity of the indirect in situ PCR/RT-PCR method.

➫ It is imperative that data banks be consulted to check that there is no homology between the sequences of the probes and the possible DNA or RNA sequences that may be present in the cell.

The specificity criterion only concerns the nature of the probes. 6.3.2.3.1 OLIGONUCLEOTIDE PROBES As is the case for primers, the synthesis of probes must satisfy certain criteria: • Length

• Sequence

• Composition

• Melting temperature (Tm)

• Position

• Checking

➫ It must be between 20 and 30 mers. ➫ Small nucleotide fragments may hybridize in a nonspecific way. HPLC (high-performance liquid chromatography) purification is recommended. ➫ Being complementary to one of the strands of the amplified product, a probe must not contain any palindromic sequences or interprobe complementarity, if nonspecific hybridizations are to be avoided. ➫ The two probes must contain very similar percentage of GC. In any case it should be <55%. ➫ See Section 4.3.1.3. ➫ This is similar for the different probes. A difference of >5°C could favor the nonspecific hybridization of one of the probes. ➫ The position should be internal to the amplified product. ➫ It should not overlap the primers. The presence of primers in the cells after the washing step could result in a nonspecific signal. ➫ Each probe should be checked against a genomic data bank by analysis and comparison.

6.3.2.3.2 CDNA PROBES The cDNA sequence must be included in that of the amplified product. This cDNA can be obtained by: • Amplification after insertion into a vector, or • Synthesis by amplification of the amplified product

126

➫ This method takes a long time, and is seldom, if ever, used. ➫ It is easy to obtain the amplified product by PCR in the liquid phase, then to use a nested PCR to amplify a part of it, which can then be used as a cDNA probe.

6.3

Tools: The Probes

6.3.3 Labeling the Probes To detect the hybrid, the probe carries a label that can be revealed. The labeling method depends on the nature of the probe: • Oligonucleotide

• cDNA

• cRNA

➫ By 3′ extension. This is the most commonly used method. ➫ By 5′ extension (see Section 5.3.2.7). This method, using a radioactive nucleotide in the γ position, is rare, although it can be used to quantify amplification. With a single radioactive atom attached to the 5′ end, the emitted radiation is proportional to the number of hybrids formed, and thus the number of copies obtained. ➫ By random priming or nick translation, if the cDNA is obtained after the insertion of a plasmid. ➫ By PCR with labeled primers and, more particularly, by nested PCR if the amplified product was also obtained by PCR in the liquid phase. ➫ By in vitro transcription. This method is not used after in situ PCR/RT-PCR.

Only the two most generally used labeling methods will be presented here, namely: • Labeling by PCR • By 3′ extension The labels are: • Antigens, or • Radioactive isotopes 6.3.3.1

➫ Carried by nucleotides ➫ Biotin, digoxigenin, or fluorescein 35 33 ➫ Generally S or P

Antigenic labels

These are carried by dUTP, and are the most widely used labels.

➫ See Section 5.3.1.3.1.

❑ Advantages • Stability • Easy to use • Rapid detection process • Resolution

➫ Easy to store the labeled nucleotides and probes ➫ No radioprotection measures necessary ➫ Numerous detection methods ➫ Immunohistochemical reaction ➫ Cellular

127

Hybridization ❑ Disadvantages • There is a limit to the number of nucleotides that can be incorporated during labeling.

• The labeling of the probe needs to be checked. • Hybridization is limited. • The quantification of the signal is a delicate operation. 6.3.3.2

➫ Without precautions (e.g., the addition of unlabeled nucleotides), only one or two labeled nucleotides can be incorporated. This type of mixed extension can be extremely long, i.e., up to several times that of the probe itself. ➫ Except for probes whose fluorescent labeling can be observed directly, this check requires a stained reaction (see Section 6.3.3.6). ➫ This is due to the steric size. ➫ Stained reactions, using a chromogen, are not quantifiable.

Radioactive labels

The two main ones are: • •

35 33

➫ See Figure 6.10.

S P

The isotope is substituted in the α position of the nucleotide. 35

6.3.3.2.1 S Its characteristics are: • Half-life: 87.4 days • Emission energy: 0.167 MeV • Resolution: 10 to 15 µm • Sensitivity: Medium

➫ Only the phosphate in the α position is incorporated into the polymer. ➫ This is the most commonly used label. ➫ This is long enough for the probe to be used before radiolysis occurs. − ➫ Emission of β particles at a level similar 33 to that of P, allowing good localization of the signal. 33 ➫ Similar to that given by P 33 ➫ Similar to that given by P ➫ Specific activity: 1500 Ci/mmol

• Autoradiographic efficiency: − 0.5 grain/β emission ❑ Advantages • A less energetic isotope • An excellent compromise

➫ The handling of S does not require onerous radioprotection measures. ➫ This is between sensitivity and efficiency. 35

❑ Disadvantages • The nucleotide is modified (substitution of an oxygen molecule in the phosphate group). • There is a risk of oxidation. •

128

35

S is difficult to use.

➫ The chemical bond is unstable. ➫ It necessitates the use of protection (e.g., DTT, mercaptoethanol). ➫ It can bring about chemical modifications in the molecule, thus causing background.

6.3

Tools: The Probes

33

6.3.3.2.2 P 33 32 The P label has all the advantages of the P label, and only minor disadvantages. • • • • •

➫ ➫ ➫ ➫ ➫

Half-life: 25.4 days Emission energy: 0.25 MeV Resolution: 15 to 20 µm Sensitivity: Medium Autoradiographic efficiency

Short exposure time 35 Close to that of S Good 35 Close to that of S 35 Close to that of S

❑ Advantages ➫ Few radioprotection problems ➫ Short exposure times ➫ It is a physiological label

• Low emission energy • A short half-life • No modification of the nucleotide ❑ Disadvantages

➫ Still high ➫ Difficult to store, although quick to use

• Cost • Short half-life 6.3.3.3

Labeling by PCR

6.3.3.3.1 OVERVIEW This is a direct PCR in the liquid phase, which incorporates labeled nucleotides during the amplification step. The amplification should give a labeled probe that hybridizes in situ on a sequence of the amplified product. The probe is obtained from: • The amplified product derived from the PCR in the liquid phase, or • Genomic DNA

5’

3’ 3’

5’

3’

5’

3’

5’

➫ This method is recommended because of its specificity. ➫ This method is of no practical use, and in fact it presents the additional risk of a nonspecific attachment of the primers. ➫ PCR in the liquid phase, using the same primers as in situ PCR. ① Obtaining the amplified product

1 3’

➫ See Section 5.3.2.6. ➫ This kind of direct PCR gives a yield that is inversely proportional to the percentage of labeled nucleotides (close to 100%). ➫ The primers are situated on the sequence of the amplified product such that a smaller nested PCR fragment is generated.

5’

5’

3’ 2

② Hybridization of the primers, and extension by Taq DNA polymerase

129

Hybridization 3’

5’

5 3’

5’

3’

5’

5 5

3’

➫ Nucleotides that carry the label, and unlabeled nucleotides.

+

3 5’ 3’

➫ Each of the two primers hybridizes to one of the strands of the amplified product.

3’

③ Incorporation of the labeled nucleotides into the neosynthesized strands ➫ Double-strand probe labeled at the end of the first cycle.

5’

④ Amplification ➫ The amount of probe obtained is proportional to the number of cycles. Figure 6.2 Obtaining a probe labeled by PCR. ❑ Advantages • Specificity of the probes • Simultaneous synthesis and labeling of the two probes • Production of a large quantity of labeled probes • Labeling along the entire length of the probes • Size of the probes

➫ They are made directly from the sequence of interest. ➫ This is the basic principle of PCR. ➫ The number of cycles is the only limiting factor. ➫ The density of the labeling depends on the ratio of the labeled nucleotide to the total amount of nucleotide. ➫ It can be up to 200 or 300 bp, i.e., practically the size of the amplified product to be detected.

❑ Disadvantages • The usual difficulties involved in using radioactive nucleotides • That the two probes are entirely complementary to each other • The size of the probes 6.3.3.3.2

PROTOCOL

➫ There is the risk of contamination of the PCR apparatus. ➫ They are PCR products. In this case, there is a high level of denaturation. ➫ This can be a disadvantage if the amplified fragment is very long. ➫ For reasons of simplification, only the labeling protocol using dNTPs conjugated to an antigen is presented here.

❶ Equipment • Liquid PCR thermocycler 130

➫ Standard equipment

6.3 ❷ Reagents • Sense and anti-sense primers • dUTP-X-antigen • Unlabeled dNTPs • Taq DNA polymerase • Mineral oil • KCl • MgCl2 • Tris–HCl

➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫

❸ Solutions

• • • • • •

• Sterile water REACTION

MIXTURE

FOR

Storage in aliquots at −20°C Storage at −20°C Storage at −20°C Storage at −20°C For PCR Molecular-biology grade Molecular-biology grade Molecular-biology grade

➫ All the solutions must be prepared using DNase-free reagents in a sterile container (see Appendix A1.1). ➫ 0.1 to 1 µM (stored in aliquots at −20°C) ➫ Addition of 1.3 mM dTTP ➫ Storage at −20°C ➫ 5 U/µl (storage at −20°C) ➫ See Appendix B2.12. ➫ 100 mM Tris–HCl; 500 mM KCl; pH 8.3 ➫ Storage in aliquots at −20°C ➫ See Appendix B1.1.

Sense and anti-sense primers 0.7 mM dUTP-X-antigen 2 mM unlabeled dNTPs Taq DNA polymerase 25 mM MgCl2 10X buffer

6.3.3.3.3

Tools: The Probes

ANTIGENIC

LABELING

a. Place the following reagents in a sterile Eppendorf tube: • Amplified product X • Primers 250 nmol • dATP, dGTP, dCTP X µl • Labeled dUTP X µl • Unlabeled dTTP X µl • MgCl2 2–10 µl • 10X buffer 5 µl • Taq DNA polymerase 1.5 U • H2O b. Mix and centrifuge. c. Cover with oil. d. Place in the thermocycler. 6.3.3.3.4

To 50 µl

➫ To be determined ➫ ➫ ➫ ➫

2 mM 0.7 mM 1.3 mM To be determined

➫ To be determined (0.5 to 2.5 U) ➫ Volume according to the concentration

100 µl

PCR PROTOCOL

❶ First cycle • Denaturation • Hybridization • Extension

7 min 94°°C 1 min 60°°C 1 min 72°°C

➫ The temperature varies according to the primer. ➫ The time can be extended if the probe to be synthesized is long. 131

Hybridization ❷ Following cycles (n cycles)

➫ The number depends on the required quann tity of probe: Q = q × 2 , where Q is the final quantity of probe, q is the initial quantity of probe, and n the number of cycles.

• Denaturation

1 min 94°°C

• Hybridization

1 min 60°°C 1 min 72°°C

• Extension ❸ Last cycle • Denaturation • Hybridization • Extension

1 min 94°°C 1 min 60°°C 10 min 72°°C

➲ Following step • Precipitation with ethanol 6.3.3.4

➫ For large numbers of cycles, it is sometimes necessary to reduce the time to preserve the efficiency of the enzyme. ➫ The temperature varies according to the primer. ➫ After 10 to 20 cycles, the time is generally increased to compensate for the loss in efficiency of the enzyme.

➫ In this cycle the time needs to be longer than in the previous cycles so that the extension of the newly formed strands can be completed. ➫ See Section 6.3.3.5.

Labeling by 3′′ extension

This labeling method is used with oligonucleotides. 6.3.3.4.1 OVERVIEW The labeling of an oligonucleotide (<50 mers) is carried out by the addition of labeled nucleotides at the 3′ end using an enzyme, i.e., terminal deoxynucleotidyl transferase (TdT). This requires a free 3′ OH and nucleotide triphosphates. In the presence of cobalt ions, the enzyme (TdT) catalyzes the polymerization of a labeled deoxyribonucleotide.

132

➫ Random priming and PCR are not applicable to these oligonucleotides. ➫ In the case of antigenic nucleotides it is possible to add labeled and unlabeled nucleotides alternately, to obtain a longer extension. ➫ Cobalt is the cofactor of TdT.

6.3

Tools: The Probes

① Synthesized oligonucleotide probe (the 3′ end must be hydroxylated).

1 5’

3’

② Enzymatic action: The terminal deoxynucleotidyl transferase (TdT) attaches itself to the 3′ end, which contains a free OH.

2 TdT

5’

3’

+

③ The addition of labeled dUTP and unlabeled dATPs, and polymerization: The TdT enzyme catalyzes the polymerization of the labeled deoxynucleotide triphosphates.

3

5’

3’

Figure 6.3 Labeling by 3′′ extension.

❑ Advantages • Radioactive or antigenic labels are used. • The two probes cannot hybridize with each other.

• This is a rapid, simple labeling method. • It is possible to store the labeled probe. • Quantification is possible.

➫ Quantification is possible. ➫ The regions to be hybridized can be located anywhere on the sequence of the product to be amplified. ➫ Reassociation with the probes is not possible. ➫ Storage is possible in the case of antigenic labeling. ➫ Each hybrid corresponds to a target nucleic acid.

❑ Disadvantage • There is a possibility of the labeled probes diffusing.

6.3.3.4.2 EQUIPMENT/REAGENTS/SOLUTIONS ❶ Equipment • Incubator at 37°C ❷ Reagents • Potassium cacodylate • Cobalt chloride (CoCl2) • Antigenic dUTP labels —Biotin-X-dUTP —Digoxigenin-X-dUTP, or —Fluorescein-X-dUTP

➫ Given their small size, these probes may attach to damaged cell structures, thus increasing the risk of background.

➫ Enzymatic reaction ➫ Molecular-biology grade ➫ To be used only for in situ hybridization ➫ Labeling kit

133

Hybridization

• Oligonucleotides

• Terminal deoxynucleotidyl transferase (TdT)

❸ Solutions

• CoCl2 25 mM • Antigenic deoxynucleotides —1 mM biotin-X-dUTP —1 mM digoxigenin-X-dUTP —1 mM fluorescein-X-dUTP • Radioactive deoxynucleotides 35 33 —α( S or P)-dATP

• Water • 2 to 100 pmol/µl oligonucleotides • Labeling (tailing) 5X buffer

• 50 U/µl terminal deoxynucleotidyl transferase (TdT)

6.3.3.4.3

33

➫ All the solutions are prepared using DNase- and RNase-free reagents in a sterile container (see Appendix A1.1). ➫ Storage at −20°C (see Appendix B2.3.2) ➫ Storage at −20°C ➫ See Figure 6.4. ➫ See Figure 6.5. ➫ See Figure 6.6. 35 33 ➫ The radioisotope ( S or P) occupies the α position (see Figure 6.10) for labeling by 3′ extension. ➫ Storage in aliquots at −80°C or 4°C ➫ Specific activity ≈3000 Ci/mmol ➫ Treated with DEPC (see Appendix A1.2) ➫ For labeling ➫ 1 M potassium cacodylate; 125 mM Tris– HCl; 1.25 mg/l BSA; pH 6.6 ➫ Storage at −20°C ➫ 200 mM potassium cacodylate; 1 mM EDTA; 200 mM KCl; 0.2 mg BSA; 50% glycerol (v/v); pH 6.5 ➫ Storage at −20°C. The presence of glycerol means that the enzyme solution remains liquid at −20°C. The enzyme is labile. ➫ Labeled oligonucleotides: 35 • α S-dATP 33 • α P-dATP

PROTOCOL FOR RADIOACTIVE PROBES

❶ Reaction mixture Place the following reagents in a sterile tube in the order indicated:

134

➫ Essentially α( S or P)-dATP; other radioisotopes are little used. The use of other dNTPs can lead to nonspecific signals. ➫ 25 to 45 nucleotides (mers); 30 nucleotides is a good average. ➫ Storage is at −20°C. ➫ The enzyme is often sold with the appropriate buffer and CoCl2. 35

• Radioactive dATP labels

➫ Eppendorf type 5 µl 5 µl

• Sterile water • 10 pmol or 100 pmol oligonucleotides • 5X reaction buffer • 25 mM CoCl2

4 µl 4 µl

• 10 pmol labeled dATP • 50 U/µl TdT

1 µl 1 µl

➫ For a final volume of 20 µl ➫ According to requirements ➫ If a red precipitate forms, need to check the origin of the radioactive nucleotide 35 ➫ ≈50 µCi in the case of the α S-dATP probe ➫ A labile enzyme; must not be allowed to rise to room temperature

6.3 ❷ Incubation

❸ Stopping the reaction Incubate • In a water bath, or • By heating ➲ Following step • Precipitation with ethanol 6.3.3.4.4

60 min 37°°C

4°°C 10 min 65°°C

4 µl 5 µl

• Sterile water • 10 pmol or 100 pmol oligonucleotides • 5X reaction buffer • 25 mM CoCl2 • 1 mM labeled dUTP

4 µl 4 µl 1 µl

• 10 mM dATP

1 µl

• 50 U/µl TdT

1 µl

60 min 37°°C 4°°C

➲ Following step • Precipitation 6.3.3.5

➫ An optional step

➫ (biotin, digoxigenin, or fluorescein)-dUTP

❶ Reaction mixture Place the following reagents in a sterile tube, in the order indicated:

❸ Stopping the reaction • In a water bath

➫ The labeling is carried out at 37°C (30 min minimum). It is not necessary to prolong the incubation time, or to increase the amount of enzyme, because the 3′ extension remains limited (see Section 6.3.3.6).

➫ See Section 6.3.3.5.

PROTOCOL FOR ANTIGENIC PROBES

❷ Incubation

Tools: The Probes

➫ Eppendorf type ➫ For a final volume of 20 µl ➫ Oligonucleotides diluted in sterile water, and stored at −20°C ➫ Only one or two labeled nucleotides added at the 3′ end ➫ The addition of unlabeled dATP, which allows the incorporation of several labeled dUTPs ➫ Catalyzes the polymerization of nucleotides at the 3′ end ➫ A labile reagent, which must not be reheated ➫ Not necessary to prolong the incubation time, or to increase the amount of enzyme, given that the extension is limited ➫ An optional step, which can be replaced by the addition of EDTA (2 µl), or by heat (65°C for 10 min) ➫ The oligonucleotides are purified by ethanol precipitation (see Section 6.3.3.5).

Purification

6.3.3.5.1 OVERVIEW Nucleic acids are water soluble, and precipitate in an alcoholic solution in the presence of salts.

➫ Alternatively, nucleic acids can be separated from free nucleotides and labeling reagents by passage through a column.

135

Hybridization 6.3.3.5.2 EQUIPMENT/REAGENTS/SOLUTIONS ❶ Equipment • Centrifuge • Vacuum jar or Speedvac • Freezer (−20°C or −80°C) ❷ Solutions

• • • • •

➫ All the solutions should be prepared using DNase- and RNase-free reagents in a sterile container (see Appendix A1.1). ➫ See Appendix B2.18. ➫ See Appendix B.2.2. ➫ See Appendix B2.15. ➫ Storage at −20°C ➫ Storage at −20°C

3 M sodium acetate; pH 5.2 7.5 M ammonium acetate; pH 5.5 10 mg/ml transfer RNA (tRNA) 4 M lithium chloride Ethanol, 100° and 70°

6.3.3.5.3 PROTOCOL ❶ Reaction mixture a. On ice, add the reagents in the following order: • 10 mg/ml tRNA 2 µl • 7.5 M ammonium acetate, or • 3 M sodium acetate, or • 4 M lithium chloride

• Ethanol 100°

1/5 the final volume ≈2–3 vol

b. Vortex, centrifuge. ❷ Incubation a. Incubate.

b. Centrifuge. ❸ Washing a. Remove the supernatant. b. Wash the precipitate. • Ethanol 70° c. Centrifuge.

d. Remove the supernatant. e. Dry. 136

➫ ≥14,000 g ➫ To dry the probe ➫ To store reagents and precipitation products

60 min −80°°C or overnight −20°°C >14,000 g ≥15 min 4°°C

➫ This facilitates the precipitation of oligonucleotide probes; optional. ➫ The ammonium acetate can be replaced by 3 M sodium acetate or 4 M lithium chloride. In the latter case, the pellet must be washed with 70° alcohol (stored at −20°C) after precipitation and recentrifugation. ➫ Store at −20°C. ➫ The volume of the reaction solution ➫ No reagent must remain on the side of the tube. ➫ Precipitation of the probe ➫ Minimum 30 min at −80°C, or ➫ Minimum 2 h at −20°C ➫ With the tube oriented to facilitate the localization of the pellet and the removal of the supernatant ➫ May contain radioactive nucleotides

50 µl >14,000 g ≥15 min 4°°C

➫ To eliminate salts ➫ With the tube oriented to facilitate the localization of the pellet and the removal of the supernatant

6.3 6.3.3.6

Tools: The Probes

Checking/storage/utilization

Before a probe is used or placed in storage, its labeling must be checked. 6.3.3.6.1 CHECKING THE LABELING The type of check depends on the label: ❶ Antigenic labels ❷ Fluorescent labels ❸ Radioactive labels The length of a labeled probe can be checked by the use of an agarose gel.

6.3.3.6.2 STORAGE This depends on the nature of the label: ❶ Antigenic probes

❷ Radioactive probes

6.3.3.6.3 UTILIZATION Double-strand probes (i.e., obtained by PCR) have to be denatured before hybridization. a. Denature. 10 min 92°°C or 5 min 96°°C b. Cool quickly in ice. 0°°C c. Utilize.

➫ An immunohistochemical reaction is used to reveal a range of dilutions of the labeled probe on a nylon membrane. ➫ The detection of the fluorescence after deposition of a range of dilutions on a nylon membrane exposed to ultraviolet radiation. ➫ This is a measure of the specific activity of the probe. ➫ This check needs to be carried out only if the background level is high. The specificity of small fragments is very limited.

➫ Antigenic labels are very stable. ➫ Solubilize in Tris–EDTA buffer (see Appendix B3.6). ➫ Store at −20°C for several months. ➫ At −80°C, the risk of radiolysis is limited. ➫ The half-life of the radioelements: 33 • P: half-life 25.4 days; storage 1 week 35 • S: half-life 87.4 days; storage <1 month ➫ Store in a dry state, or in sterile water at −80°C. ➫ Oligonucleotide probes can be used directly. ➫ If the probe is labeled with S, add 10 mM DTT. 35

➫ At this temperature, the two strands cannot rehybridize. ➫ The probe can be denatured in the hybridization reaction medium.

137

Hybridization

6.4 HYBRIDIZATION PARAMETERS The specificity of the hybridization reaction depends on the factors that limit the nonspecific binding of the probes.

❶ Hybridization temperature

❷ Salinity ❸ Hybridization buffer ❹ Type of probe ❺ Probe length

➫ Specificity is very important in the indirect PCR/RT-PCR method, in that it limits the kind of stringent washing which is needed to eliminate nonspecific hybrids, but which can also damage tissue or cell structures that are already in a delicate state. ➫ The higher the temperature, the more specific the hybridization will be. Above Tm (see Section 4.3.1.3), 50% of the specific hybrids are denatured. ➫ The salt concentration is proportional to the stability of specific and nonspecific hybrids. ➫ Its pH affects the stability of the hybrid. ➫ DNA–DNA hybrids are less stable than DNA–RNA hybrids. ➫ The longer the probe, the more specific the hybrid.

6.4.1 Hybridization Temperature This is the temperature at which the hybridization reaction is carried out. The sections incubate in the presence of the reaction medium, in a moisture chamber, at the temperature where the lowest possible number of nonspecific hybrids is formed. This temperature depends on the Tm of the probes. 6.4.1.1

138

➫ Tm = the temperature at which 50% of specific hybrids are denatured (see Section 4.3.1.3).

Melting temperature (Tm)

The Tm is calculated using formulae that are precisely adapted to the nature of the probe. The calculation of the Tm can be carried out automatically by certain types of software on the basis of the probe sequences. 6.4.1.2

➫ The formation of hybrids is a spontaneous reaction.

➫ See Section 4.3.1.3. ➫ This acts as a check that the Tm of the chosen pair of probes are mutually compatible.

The difference between Tm and the hybridization temperature

The hybridization temperature is determined empirically, and depends on the nature of the probe:

➫ If the hybridization temperature is too far below the Tm, a large number of hybrids will form, but their specificity will be reduced.

❶ cDNA probe • The hybridization temperature is 10°C below the Tm. ❷ Oligonucleotide probe • The hybridization temperature is ≈5°C below the Tm.

➫ 200 to 300 bp ➫ 20 to 30 mers

6.4 The hybridization temperature can be artificially lowered by the addition of formamide. The influence of temperature on the hybridization process can be illustrated as follows:

1

100 °C Tm 37°C 0°C -

2

100 °C Tm 37°C 0°C -

3

100 °C Tm 37°C 0°C -

4

100 °C Tm 37°C 0°C -

5

100 °C Tm 37°C 0°C -

6

Between 94 and 100°°C, denaturation takes place, i.e., the two strands separate out.

If, after denaturation, the single-stranded DNA is immediately brought down to a temperature close to 0°°C, no hybrid will form. If the hybridization temperature is close to room temperature, a large number of hybrids, both specific and nonspecific, will form.

The ideal hybridization temperature, i.e., the one at which the maximum number of specific hybrids will be formed, is 5 to 10°°C below the Tm.

If the hybridization temperature is the same as the Tm, only 50% of specific hybrids will form (this follows from the definition of Tm).

Finally, if the hybridization temperature reaches 94 to 100°°C, no hybrids will form, as the nucleic acids are denatured.

100 °C Tm 37°C 0°C -

➫ The addition of 1% formamide lowers the hybridization temperature by 0.65°C.

At room temperature, and up to 37°°C, the DNA matrix is double stranded.

100 °C Tm 37°C 0°C -

Hybridization Parameters

7

Figure 6.4 Determination of the hybridization temperature. 139

Hybridization +

➫ Salinity

6.4.2 Na Ion Concentration +

The Na ion concentration influences the stability of the hybrids. If it is raised tenfold, the Tm will rise by 16.6°C, which means that the hybrids will be more stable.

➫ The most common concentration is around 660 mM. This corresponds to 4X SSC buffer (see Appendix B3.5).

6.4.3 Hybridization Buffer ➫ Large variations in the pH destabilize interbase hydrogen bonds.

This limits variations in the pH.

6.4.4 The Nature of the Probes Since DNA–RNA hybrids are more stable than DNA–DNA hybrids, it may be necessary, in very rare cases, to use RNA probes.

➫ This type of hybridization does not take place, in practice, after in situ PCR/RT-PCR.

6.4.5 Probe Length The longer the probe, the higher the specificity and stability of the hybrids. Labeling by the 3′ extension of an oligonucleotide probe can give a probe that is two to three times longer than the original probe.

➫ The Tm rises with the length of the probe. ➫ This fact must be taken into account when calculating the hybridization temperature.

6.4.6 Duration Most authors agree that the reaction is optimal after 3 h of incubation.

3–16 h

➫ The reaction can be continued for up to 16 h (i.e., overnight) without any risk for the hybrids. Morphology, however, deteriorates over time.

6.5 PROTOCOL 6.5.1 Equipment/Solutions 6.5.1.1

Equipment

• Cover slides — 12 × 12 mm cover slides — 24 × 50 mm cover slides — 9 mm ∅ circular cover slides • Moisture chamber

140

➫ Sterilized, wrapped in aluminum foil, and stored at room temperature; gloves must be worn when handling them ➫ Of the petri box type (≈24 × 24 cm), with enough space for the slides

6.5 • Oven • Thermocycler • Vortex mixer 6.5.1.2

Protocol

➫ 37 to 65°C ➫ Can also be used for the hybridization step

Solutions

• Alcohol 70°, 95°, and 100° • Deionized formamide

• 50X Denhardt’s solution

• 50% dextran sulfate • 1 M dithiothreitiol

• 10 mg/ml DNA

• 0.5 mg/ml heparin • 10 mg/ml poly (A) • 10 mg/ml RNA

• Sarcosyl • 20X SSC (standard saline citrate), pH 7.0 • Sterilized water

• TE buffer/NaCl

➫ See Appendix B2.4. ➫ The formamide must be of good quality (very pure) and deionized (to maintain the pH of the buffer). ➫ Storage is at −20°C in aliquots of 500 µl. If the reagent is not frozen at −20°C, it should not be used. ➫ See Appendix B2.5. ➫ Storage is at −20°C in aliquots. Unfreezing several times is possible without modification of its properties. ➫ See Appendix B2.6. ➫ Storage is at −20°C. ➫ See Appendix B2.7. ➫ Storage is at −20°C in aliquots of 50 to 100 µl. It cannot be refrozen. Its nauseous odor is the best guarantee of its condition. It should not be used if the odor is modified or absent. ➫ See Appendix B2.8. ➫ Sonication ➫ Storage is at −20°C. The freezing/unfreezing cycles can cause breaks. ➫ Storage is at −20°C. ➫ See Appendix B2.13; equivalent to RNA. ➫ See Appendix B2.15. ➫ Storage is at −20°C. The freezing/unfreezing cycles can cause breaks. ➫ See Appendix B2.17. ➫ See Appendix B3.5. ➫ Storage is at 4°C, or at room temperature. ➫ See Appendix B1.1. Use only immediately upon opening the bottle, or use DEPC-treated water (see Appendix B1.2). ➫ See Appendix B3.6.2.

6.5.2 The Reaction Medium The reaction medium is made up of the hybridization buffer plus the two probes.

➫ The hybridization buffer can be stored, at least for a few weeks, at −20°C, either alone or added to the probe, without losing any of its effectiveness.

141

Hybridization ❶ Hybridization buffer • 20X SSC buffer

× 4×

• Deionized formamide

50%

• 50% dextran sulfate, or

10%

• 0.5 mg/ml heparin • 10 mg/ml RNA, or

0.1 mg/ml 100–250 µg/ml 100 µg/ml

• 10 mg/ml poly (A) • 10 mg/ml DNA • 50X Denhardt’s solution • 1 M DTT

100–400 µg/ml 1–2X 10 mM

❷ Reaction medium a. Add the probe to the hybridization buffer.

• Radioactive probe

0.1–0.2 µg/ml hybridization buffer

• Antigenic probe

1–10 µg/ml hybridization buffer

b. Vortex. c. Centrifuge.

6.5.3 The Different Steps a. Place the reaction medium on the tissue sections. 142

30–50 µl/ section

➫ Hybrids form at pH close to neutrality in the + presence of Na ions (ionic strength). + ➫ The Na ion concentration is of major significance for the stability of the hybrid, on which it has a direct effect. ➫ This is Indispensable. ➫ The addition of formamide reduces the hybridization temperature (see Appendix B2.4). ➫ This increases the effectiveness of the hybridization process by lowering the Tm. ➫ This increases the effectiveness of the hybridization reaction, and also speeds it. It is a nonionic polymer that concentrates the solute through the volume that it occupies in the solution. ➫ This protects cellular RNA. ➫ This can replace dextran sulfate. ➫ This saturates nonspecific bonds of the proteic type by competition. ➫ This saturates nonspecific bonds; can be replaced by a poly (C) or a poly (G). ➫ This saturates nonspecific bonds. ➫ This saturates nonspecific bonds by competition with macromolecules. ➫ This is used only with probes labeled with 35 S (it is an antioxidant against the radiolysis 35 of S). ➫ This is used only after denaturation (3 min at 100°°C), since it is destroyed at 100°°C. ➫ Add up to the “saturating” concentration, i.e., the concentration at which no further addition of probe will modify the intensity of the signal. ➫ After centrifugation and drying, the labeled probe is dissolved directly in the hybridization buffer.

6.6 b. Cover the sections with: • cover slides, or • “Cover clips” c. Denature.

3 min 100°°C d. Place the slides directly in the moisture chamber.

e. Incubate.

At least 3 h, and up to 16 h

Posthybridization Treatments

➫ If the sections are hybridized in a moisture chamber ➫ If the sections are hybridized in a thermocycler ➫ Indispensable, since the amplification product is double-stranded DNA ➫ With 5X SSC buffer, in the incubator programmed for the chosen temperature. ➫ Possible also to program the thermocycler for this step ➫ More generally, overnight

6.6 POSTHYBRIDIZATION TREATMENTS 6.6.1 Aim The aim is to eliminate as many nonspecific hybrids as possible, while conserving the maximum number of specific hybrids. During the hybridization step, the probes fix with a very high degree of affinity to perfectly homologous nucleic acid molecules, but also, with a lesser degree of affinity, to nucleic sequences whose homology is only partial. The purpose of posthybridization treatments (e.g., washes) is to eliminate nonspecific hybridization, which introduces nonspecific signals and masks specific hybridization by reducing the signal to noise ratio.

➫ Matched hybrid ➫ Mismatched hybrid ➫ A succession of washes is carried out in increasingly stringent conditions so that only specific hybrids, which are the most stable, remain. ➫ Furthermore, a probe tends to fix in a totally nonspecific way to non-nucleic structures, essentially those of proteins.

6.6.2 Parameters The parameters that determine the degree of stringency, and thus the choice of hybrids (in terms of their stability), are: ❶ Temperature ❷ Ionic strength ❸ Washing time The more stringent the conditions, the more the hybrids are denatured.

➫ Mismatched hybrids are denatured before matched hybrids.

143

Hybridization 6.6.2.1

➫ This is the most important factor for the stability of hybrids.

Temperature

The higher the temperature, the higher the risk of denaturation. Only internal control makes it possible to determine the optimal washing temperature. The presence of negative and positive cells confirms the disappearance of nonspecific hybrids. 6.6.2.2

+

Na ion concentration +

The salt [Na ] concentration is proportional to the stability of the hybrids. Washing in decreas+ ing concentrations of Na ions brings about the dissociation of the hybrids; the less stable are denatured before the more stable. 6.6.2.3

➫ This means the disappearance of specific hybrids. ➫ For in situ hybridization, the washing temperature (Tw) is determined by washing with 2X SSC at temperatures varying by 5°C between 40 and 70°C.

➫ Mismatched hybrids and nucleic acid– protein bonds are less stable than specific hybrids. ➫ Washing in water would bring about complete denaturation.

Washing time

Little difference is observable after an hour.

Ceteris paribus, repeated washes are more effective than long washes.

➫ Washing with moderate shaking gives the best reproducibility of results. ➫ Washing accompanied by intense or prolonged shaking can cause damage to the sections, and is more difficult to reproduce. ➫ Limiting the number of washes can be compensated for by voluminous washes accompanied by shaking.

6.6.3 Protocol • 5X SSC

5 min

• 2X SSC

30 min Tw 30 min rt 30 min rt 30 min rt

• 2X SSC • 1X SSC • 0.5X SSC

• 0.1X SSC

144

30 min rt

➫ For the first rinse, which serves only to unstick the cover slides, place the slides in a borrel tube or tray containing the solution, and let the cover slide off gently without touching it. ➫ Necessary ➫ Washing temperatures, to be determined ➫ Room Temperature ➫ An optional step. Temperature is a factor + of stringency that is very effective at this Na concentration. Use a water bath with shaking. ➫ An optional step. To be used only with + radioactive probes. The Na concentration is very low, and can cause dissociation of specific hybrids.

6.7

Before Revelation

6.7 BEFORE REVELATION Depending on whether the label is ➫ At this stage, the probe label is the hybrid label.

• Radioactive or • Antigenic the preparation will be different.

➫ The sections must be dried before contact with autoradiographic film or emulsion.

6.7.1 Radioactive Hybrids 6.7.1.1

Dehydration

• Alcohol 70°

1 rapid

• Alcohol 95° • Alcohol 100°

1 rapid 1 rapid

6.7.1.2

➫ Indispensable

Drying

• In a vacuum jar

➫ It is advisable to make up the solutions of alcohol with 300 mM ammonium acetate to maintain the ionic strength. This salt is volatile, so it does not cause deposits during the drying of the sections.

30–60 min

➫ The aim of this step is to eliminate all trace of alcohol and water (especially from tissue), which could cause background noise on macroautoradiograms.

➲ Following steps • Contact with autoradiographic film • Dipping in the autoradiographic emulsion

➫ See Section 7.3.3. ➫ See Section 7.3.4.

6.7.2 Antigenic labels For immunohistological detection, do not dry tissue sections. The tissue is transferred to the buffer that will be used during the detection process. ➲ Following step • Immunohistochemical detection

➫ Indispensable

➫ See Section 7.2.

145

Chapter 7 Revelation

Contents

CONTENTS 7.1

Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

151

7.2

Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

152

7.2.1

Immunohistochemical Reaction . . . . . . . . . . . . . . . . . . . . . . 7.2.1.1 Immunocytochemical Tools . . . . . . . . . . . . . . . . . 7.2.1.1.1 Antibodies . . . . . . . . . . . . . . . . . . . . . . 7.2.1.1.2 Streptavidin . . . . . . . . . . . . . . . . . . . . . 7.2.1.1.3 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1.2 Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1.2.1 Enzymes . . . . . . . . . . . . . . . . . . . . . . . 7.2.1.2.2 Fluorescent Labels . . . . . . . . . . . . . . . 7.2.1.2.3 Particle Labels. . . . . . . . . . . . . . . . . . . 7.2.1.2.4 Conjugates. . . . . . . . . . . . . . . . . . . . . . Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.1 Direct Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.1.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 7.2.2.1.2 Advantages/Disadvantages . . . . . . . . . 7.2.2.2 Indirect Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.2.1 Overview. . . . . . . . . . . . . . . . . . . . . . . 7.2.2.2.2 Advantages/Disadvantages . . . . . . . . . 7.2.2.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.3.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.3.2 Direct Reaction Protocol. . . . . . . . . . . 7.2.2.3.3 Indirect Reaction Protocol . . . . . . . . . 7.2.2.3.4 Particular Case: The Biotin Label . . . . . . . . . . . . . . . . . Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3.2 Alkaline Phosphatase . . . . . . . . . . . . . . . . . . . . . . 7.2.3.3 Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

152 152 152 153 153 154 154 155 155 155 156 156 156 156 156 156 157 157 157 158 158

7.3 Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

165

7.2.2

7.2.3

7.3.1 7.3.2

7.3.3

Principles of Autoradiography . . . . . . . . . . . . . . . . . . . . . . . Characteristics of Emulsions . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.1 Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.2 Photographic Emulsions . . . . . . . . . . . . . . . . . . . . 7.3.2.3 Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.4 Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.5 Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.6 Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.7 Artifacts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.8 Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . Macro-Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.3.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.3.2 Autoradiographic Film . . . . . . . . . . . . . . . . . . . . .

159 161 161 161 163

165 166 166 166 166 166 167 167 167 167 168 168 169 149

Revelation 7.3.3.3

7.3.4

150

Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.3.3.1 Equipment/Solutions. . . . . . . . . . . . . . 7.3.3.3.2 The Different Steps . . . . . . . . . . . . . . . Micro-Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.4.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.4.2 Types of Emulsion . . . . . . . . . . . . . . . . . . . . . . . . 7.3.4.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . 7.3.4.4 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.4.4.1 Equipment/Reagents/Solutions . . . . . . 7.3.4.4.2 The Different Steps . . . . . . . . . . . . . . .

169 169 170 171 171 171 171 172 172 173

7.1

Diagram of the Different Steps

The choice of detection method depends on the hybrid label: ➫ Detection of radiation ➫ Immunological detection

• Radioactive isotope • Antigen There are two possible approaches, depending on the label: • Immunohistochemistry

• Autoradiography

Given the amplification necessary for a strong signal, whose interpretation and validity are sometimes questionable, preference is given to detection systems that increase the signal/background ratio and give good cellular resolution.

➫ Immunohistochemistry involves an antigen– antibody reaction, which is visualized by: • A stained reaction, or • Fluorescence ➫ Autoradiography involves the visualization of emitted radiation by the use of a photographic emulsion, either: • A solid emulsion on film, or • A liquid emulsion, with which the tissue is coated ➫ What is sought is the cellular or subcellular location of DNA or RNA. Radioactive labeling does not provide enough accuracy, which is the reason antigenic labeling is much more highly favored.

7.1 DIAGRAM OF THE DIFFERENT STEPS ❶ To detect an antigenic hybrid, which comes from: • The incorporation of a nucleotide coupled to an antigenic molecule, or • A PCR or RT-PCR in the presence of a primer labeled with an antigenic molecule, or • The hybridization of the amplified product by probes into which a haptene has been incorporated.

➫ During a direct PCR or RT-PCR ➫ See Section 5.3.1.3 ➫ See Section 5.3.2.7 ➫ See Section 6.3.3

Antigenic hybrids

Direct detection

Indirect detection

151

Revelation ❷ To detect a radioactive hybrid, which comes from: • The incorporation of a radioactive nucleotide in a direct PCR, or • Amplification in the presence of a radiolabeled primer, or • The hybridization of the amplified product by radioactive probes

➫ α[ S]dATP or dCTP ➫ This method is not recommended (see Section 5.3.1.3) 35 ➫ γ[ S]dATP or dCTP (see Section 5.3.2.7) 35

Radioactive hybrids

Macro-auto radiography

Micro-auto radiography

7.2 IMMUNOHISTOCHEMISTRY The antigenic hybrid has one or more sites (i.e., hapten) where an antigen–antibody complex can form during the immunohistological reaction.

➫ The most commonly used haptenes are: • Biotin • Digoxigenin • Fluorescein (see Section 5.3.1.3.1)

7.2.1 Immunohistochemical Reaction The purpose of this reaction is to form a complex of high affinity between the antigen and a molecule that is specific to it (a tool) to visualize the antigen, and thus the amplified products. 7.2.1.1

Immunocytochemical tools

These are molecules which have a high degree of affinity with haptenes. • Antibody • Streptavidin

7.2.1.1.1

ANTIBODIES

• Immunoglobulin of polyclonal or monoclonal origin • Fragments of IgG: Fab and F(ab′)2 152

➫ Such molecules are produced as a response to immunization by the haptene. ➫ This spontaneously forms a complex with biotin.

➫ It is the G immunoglobulins (IgG) that are most widely used in immunohistology.

7.2

Immunohistochemistry

➫ The F(ab′)2 immunoglobulin fragment is obtained by enzymatic digestion (cleavage by pepsin) (variable sequence of Mw = 100 kDa), which, after reduction, can give rise to two Fab′ fragments with properties analogous to those of the Fab fragment. ➫ The Fab immunoglobulin fragment is obtained by enzymatic digestion (action of papain) (variable sequence). ➫ The Fc immunoglobulin fragment is obtained by enzymatic digestion (action of pepsin) (constant sequence). Figure 7.1 Molecule of IgG, and fragments resulting from its proteolytic digestion. 7.2.1.1.2 STREPTAVIDIN 15 Streptavidin has a very high affinity (Kd = 10 −1 Μ ) for biotin. One molecule of streptavidin can bind four biotin molecules by noncovalent binding.

➫ This is a homotetramer isolated from Streptomyces avidinii. ➫ In comparison to avidin, its advantages are that: • It is a nonglycosylated protein. • It has a neutral isoelectric point. • It does not interact with lectins. ➫ Similar molecules are available commercially, e.g., avidin and streptavidin: • Avidin: a glycoprotein of low molecular mass obtained from egg white, which has four fixation sites for biotin • Extravidin

Figure 7.2 Streptavidin–biotin complex.

7.2.1.1.3 BIOTIN One of the properties of this vitamin is its very high affinity bond with streptavidin (see Figure 6.4).

➫ Biotin is used as: • A haptene (i.e., labeled nucleotides; see Section 5.3.1.3) • A conjugated label (see Section 7.2.1.1) 153

Revelation It can be coupled with: • IgG • Fab, F(ab′)2 fragments • Streptavidin 7.2.1.2

➫ In this case it is not conjugated to a label, and must be considered as an antigen. ➫ Unsaturated complex.

Labels

Different types of label can be used:

➫ These labels are adsorbed onto either immunoglobulin or streptavidin.

• Enzymatic • Fluorescent • Particular 7.2.1.2.1 ENZYMES Some enzymes are more stable, and for this reason are more commonly used. ❶ Peroxidase Horseradish peroxidase (black radish extract) is an enzyme of Mw = 40 kDa.

➫ Numerous chromogens exist for all the different enzymes. ➫ This is a small molecule.

❑ Advantages • Numerous chromogens • Multiple labeling • Insoluble in alcohol

➫ Precipitates of different colors (see Section 7.2.3) ➫ Complementary to alkaline phosphatase ➫ The possibility of embedding in resin

❑ Disadvantages • Endogenous enzymes • Diffusion of the precipitate ❷ Alkaline phosphatase Alkaline phosphatase (extracted from calf intestine) is a very widely used enzyme of Mw = 80 kDa.

➫ Pretreatment of sections by the addition of an inhibitory agent such as 10% hydrogen peroxide, combined or not with sodium nitrite or methanol (see Appendix B6.2.2) ➫ This is a large molecule. ➫ This enzyme is frequently found in biological tissue. Its endogenous activity is inhibited by levamisole or heat (see Appendix B6.2.1.2).

❑ Advantages • Numerous chromogenic substrates • Sensitivity

• Simplicity, reproducibility

• Multiple labeling • Ease of observation

154

➫ Precipitates of different colors are available. ➫ The NBT-BCIP (see Appendix 6.1) system produces two precipitates with a single alkaline phosphatase molecule. ➫ Ready-to-use chromogenic systems are available. ➫ The conjugates are available in all forms (IgG, Fab, streptavidin, etc.). ➫ There is complementarity to peroxidase. ➫ Bright-field microscopy is used.

7.2

Immunohistochemistry

❑ Disadvantages • Soluble in alcohol

• Cannot be conserved • Endogenous phosphatase • Resolution less than that obtained with fluorescent labels 7.2.1.2.2 FLUORESCENT LABELS A fluorochrome is a molecule whose structure includes conjugated double bonds that, in response to excitation by a photon, emit another photon of a longer wavelength than that of the exciting photon. These labels are little used for detection purposes because of their low level of resolution and the lack of sensitivity of the methods associated with them.

7.2.1.2.3 PARTICLE LABELS Particles of colloidal gold can also be used, but their size means that latensification with silver is necessary if they are to be observed by light microscopy. 7.2.1.2.4

CONJUGATES

❶ Conjugates-antibodies • Immunoglobulin, or • Fragments can be conjugated with all the aforementioned labels.

❷ Streptavidin conjugates The same conjugates are obtained with streptavidin. ❸ Biotin conjugates The low molecular mass of biotin means that several molecules can bind to each molecule of the label.

➫ This avoids dehydration. ➫ Embedding after dehydration is possible if the reaction is very intense (loss of signal, but also attenuation of the background). ➫ Chromogens are unstable over time. Conservation is possible at 4°C. ➫ Pretreatment is indispensable to the inhibition of the signal (see Appendix B6.1.2). ➫ Colored precipitates are diffused around enzymatic sites. ➫ This structural property is responsible for the fluorescence resulting from excitation by light. ➫ Observation necessitates a fluorescence or confocal microscope. ➫ The signal cannot be conserved, in spite of any precautions that may be taken (antifading techniques, darkness, storage of slides at 4°C). ➫ 1 to 20 nm ➫ See Section 8.13

➫ Because labeling is difficult to reproduce, it is better to use commercially available products. ➫ The experimental conditions for the preparation of labels, and their adsorption, necessitate specific conditions for each label and each tool. ➫ According to its size, a label can form a complex either with one or several immunoglobulins, or with their fragments.

➫ All the biotin conjugates are commercially available.

155

Revelation ❹ Biotin–streptavidin conjugates The concentration of conjugated biotin must not be saturating for the streptavidin–biotin binding sites.

➫ See Figure 7.5.

7.2.2 Detection The detection of an antigen contained in the amplified products (direct PCR/RT-PCR reaction) or the hybrids (indirect PCR/RT-PCR reaction) is obtained by an immunohistochemical reaction, either:

➫ See Chapter 1.

• Direct, or • Indirect 7.2.2.1

Direct reaction

7.2.2.1.1 OVERVIEW The antigen is detected by a primary antibody (IgG), or fragments conjugated to a label. The haptene incorporated into the hybrid is detected by: ① Fab fragments conjugated to a label ② Immunoglobulins (primary antibodies) conjugated to a label

Figure 7.3 The direct immunohistochemical reaction. 7.2.2.1.2

ADVANTAGES/DISADVANTAGES

❑ Advantages • Rapidity • The possibility of amplification

➫ A single step ➫ Which is nonetheless limited

❑ Disadvantage • Low sensitivity

7.2.2.2

Indirect reaction

7.2.2.2.1 OVERVIEW The antigen is detected by a two-step reaction involving:

156

➫ Essentially due to steric hindrance, and to the low level of antigenic labeling of the probes

➫ The possibility of amplification

7.2

Immunohistochemistry

• The formation of a complex with haptene by a succession of antigen–antibody reactions, and • A conjugated molecule antibody (IgG), or fragments conjugated to a label. ① The haptene incorporated into the hybrid is detected ② By an anti-haptene derived from species X [IgG, Fab, F(ab)′′2], then ③ By an anti-species-X [IgG, Fab, F(ab)′′2], conjugated to a label Figure 7.4 The indirect immunohistochemical reaction. 7.2.2.2.2

ADVANTAGES/DISADVANTAGES

❑ Advantages • Sensitivity > the direct method • Amplification of the signal

➫ Several secondary antibodies can fix to the primary antibody. ➫ There is a cascade of antigen–antibody reactions.

❑ Disadvantage • Long reaction time

7.2.2.3 7.2.2.3.1

➫ There are successive incubations with each component.

Protocol SOLUTIONS

• Antibodies • Nonconjugated anti-haptene IgG • IgG, F(ab′)2, Fab — Conjugated anti-haptene — Anti-species X conjugate • Inhibition of endogenous enzymatic activity — Phosphatases — Peroxidases • Buffers — Blocking buffers 50 mM Tris–HCl buffer; 300 mM NaCl; 1% albumin serum 50 mM Tris–HCl buffer; 300 mM NaCl; 2% goat serum — 50 mM Tris–HCl buffer, pH 7.6

➫ ➫ ➫ ➫ ➫

Indirect reaction Monoclonal or polyclonal IgG Possible to use any label Direct reaction Indirect reaction

➫ See Appendix B6.2.1.2. ➫ See Appendix B6.2.1.3. ➫ See Appendix B6.2.1.1. ➫ Other agents can be added to the blocking buffer (see Appendix B6.2.1.1). ➫ For Tris–HCl, see Appendix B3.7.1. 157

Revelation — 50 mM Tris–HCl buffer; 300 mM NaCl, pH 7.6 7.2.2.3.2

➫ All the different steps are carried out at room temperature.

DIRECT REACTION PROTOCOL

a. Rinse. • Tris–HCl/NaCl buffer b. Block the nonspecific sites. • Blocking buffer

10 min

➫ This balances the osmolarity of the tissue after the posthybridization washing steps.

15–30 min

➫ Indispensable for eliminating any reaction at nonspecific sites, as the sections are preincubated with a nonspecific serum ➫ Either by aspiration or by carefully wiping the part of the slide around the tissue with filter paper ➫ Optional (see Appendix B6.2.1); the presence of endogenous enzymatic activity must be checked ➫ Formation of the haptene–antibody complex ➫ Primary antibody: (IgG), conjugated with Fab fragments (see Section 7.2.1.2) ➫ Dilution of the antibody always weak (1:10 to 1:100, according to the manufacturer’s instructions) ➫ Moisture chamber (water on filter paper)

c. Eliminate excess buffer.

d. Inhibit endogenous enzyme.

e. Spread the conjugated antibody. • Diluted in Tris–HCl/NaCl buffer

f. Incubate the antibody. g. Rinse. • Tris–HCl/NaCl buffer ➲ Following steps • Detection • Observation 7.2.2.3.3

≥20 µl/ section

2 h– overnight 3 × 10 min

➫ Can be increased if background ➫ See Section 7.2.3. ➫ See Chapter 11. ➫ All the following steps are carried out at room temperature. It is possible to make a circle of hydrophobic material to limit the quantity of solution spread on the section.

INDIRECT REACTION PROTOCOL

a. Rinse • Tris–HCl/NaCl buffer

10 min

➫ This balances the osmolarity of the tissue after the posthybridization washing steps.

b. Block nonspecific sites. • Blocking buffer

15–30 min

➫ This is an optional step for limiting any reaction at nonspecific sites. The sections are preincubated with a nonspecific serum. ➫ Optional (see Appendix B6.2.1). The presence of endogenous enzymatic activity must be checked.

c. Inhibit endogenous enzymatic activity.

158

➫ For Tris–HCl/NaCl, see Appendix B3.7.5.

7.2 d. Rinse • Tris–HCl/NaCl buffer e. Eliminate excess buffer.

3 × 10 min

f. Spread the antibody. ≥20 µl/ • Diluted in Tris–HCl/NaCl buffer section g. Incubate the antibody. 60–90 min h. Rinse • Tris–HCl/NaCl buffer 3 × 10 min i. Eliminate excess buffer. j. Spread the conjugated antibody. ≥20 µl/ • Diluted in Tris–HCl/NaCl buffer section

k. Incubate the antibody l. Rinse. • Tris–HCl/NaCl buffer m. Eliminate excess buffer.

➫ There is a risk of dilution during the following step. ➫ This forms the haptene–antibody complex. ➫ Dilute to 1:50 to 1:500 (according to the manufacturer’s instructions). ➫ Moisture chamber (water on filter paper) ➫ Or Tris–HCl buffer

60–90 min

➫ Detection of the complex ➫ Secondary antibody: IgG, Fab-fragments, or biotin conjugates, diluted to 1:25 to 1:50 (according to the manufacturer’s instructions) ➫ Dilution less than that of the first antibody ➫ Moisture chamber (water on filter paper)

3 × 10 min

➫ On sections (≥100 µl) or in trays

➲ Following steps • Detection • Observation 7.2.2.3.4

Immunohistochemistry

➫ See Section 7.2.3. ➫ See Chapter 11.

PARTICULAR CASE: THE BIOTIN LABEL ① Direct reaction Streptavidin is conjugated to a label.

② Indirect reaction The first step uses native streptavidin, and in the second, the sites that are free of streptavidin are saturated with conjugated biotin. Conjugated streptavidin–biotin complexes are commercially available.

③ Direct or indirect immunohistological reaction The reaction uses an anti-biotin IgG. This property is used to amplify the signal in the indirect reaction. Figure 7.5 The detection of biotin. 159

7.2

Immunohistochemistry

❑ Advantages • Sensitivity • Specificity • Numerous labels available

➫ Due to the high affinity of streptavidin for biotin ➫ Multiple labelings

❑ Disadvantage • Endogenous biotin

➫ This is present in some types of animal tissue (kidney, heart, muscle, liver), and must be inhibited.

❑ Protocol ❶ Solutions • Antibodies — Anti-biotin IgG

— Antispecies conjugated IgG — Goat serum • Streptavidin • Conjugated streptavidin–biotin complex

• Inhibition of endogenous enzymatic activities: — phosphatases — peroxidases • Buffers — blocking buffers +50 mM Tris–HCl buffer; 300 mM NaCl; 1% albumin serum +50 mM Tris–HCl buffer; 2% goat serum; 0.1% Triton X-100 — 50 mM Tris–HCl buffer, pH 7.6 — 50 mM Tris–HCl buffer; 300 mM NaCl; pH 7.6 ❷ Streptavidin protocol a. Block nonspecific sites. • Blocking buffer 10 min b. Form the biotin–streptavidin complex. • Conjugated streptavidin 100 µl/ diluted to 1:20 to 1:50 in section Tris–HCl/NaCl buffer 60–90 min

➫ Conjugated (direct immunohistochemical reaction) or nonconjugated (indirect immunohistochemical reaction) ➫ See Figure 7.4, indirect immunohistochemical reaction ➫ A nonspecific antibody that can be replaced by a blocking solution ➫ Conjugated or nonconjugated (see Figure 7.5) ➫ Possible to produce this complex in two steps on the section (see Figure 7.5), using: • Nonconjugated streptavidin • Conjugated biotin ➫ See Appendix B6.2.1.2. ➫ See Appendix B6.2.1.3. ➫ See Appendix B3. ➫ See Appendix B62.1.1. ➫ The purpose of the high concentration of + Na ions is to preserve the hybrids. ➫ It is possible to add other agents to the blocking solution. ➫ For Tris–HCl buffer, see Appendix B3.7.1. ➫ For Tris–HCl/NaCl buffer, see Appendix B3.7.5.

➫ The dilution depends essentially on the label used. ➫ Streptavidin can be replaced by an antibiotin–IgG conjugate. 160

7.2

Immunohistochemistry

➫ A change of buffer is sometimes advisable.

c. Rinse. • Tris–HCl/NaCl buffer ❸ Indirect reaction protocol a. Block nonspecific sites. • Blocking buffer

20 min

10 min

b. Form the biotin–streptavidin complex. • Streptavidin 1:50 in Tris– 100 µl/ HCl/NaCl buffer section 60–90 min c. Rinse. • Tris–HCl/NaCl buffer 20 min d. Incubate the conjugated biotin • Diluted to 1:20 to 1:50 in Tris–HCl buffer e. Rinse. • Tris–HCl buffer

➫ An inhibition step for endogenous biotins may be carried out immediately before incubation with streptavidin. ➫ The dilution depends essentially on the label used. ➫ This balances the osmolarity of the tissue after the posthybridization washing steps.

60 min

➫ The dilution depends essentially on the label.

20 min

➫ A change of buffer is sometimes necessary for the detection step.

➲ Following step • Detection

➫ See Section 7.2.3.

7.2.3 Revelation Enzymes require a further step to be observed by light microscopy. 7.2.3.1

Overview

Enzymatic activity is revealed by stained reactions that can use different chromogens, depending on the desired color of the precipitate.

7.2.3.2

➫ The conjugated label

➫ Essentially alkaline phosphatase and peroxidase ➫ The activity of the enzyme catalyzes the precipitation of the chromogen as a colored substance deposited at the reaction site.

Alkaline phosphatase

The chromogens (substrates) that are most widely used with alkaline phosphatase are: ➫ See Appendix B6.2.1.1.

• NBT-BCIP • Fast-Red ❶ NBT-BCIP

➫ These two substrates are soluble in dimethylformamide. ➫ C40H30Cl2N10O6 ➫ Mw = 817.70

• NBT (nitroblue tetrazolium) N

N

N

N

NO2 O2N

N

N

N

N

C

C

OCH3

+ 2HCl

H3CO

Figure 7.6 The chemical formula of NBT. 161

Revelation • BCIP (5-bromo-4-chloro-3-indolyl phosphate)

➫ C8H6NO4BrCIPxC7H9N ➫ Mw = 433.60

O

Cl O

P

O−Na+

Br O−Na+ N H

Figure 7.7 The chemical formula of BCIP.

❑ Advantages • Sensitivity • Compatibility with multiple labeling

➫ Due to the formation of two precipitates

❑ Disadvantages • The reaction sometimes takes a long time. • The precipitate is soluble in alcohol.

➫ The detection process can take several hours. ➫ If the labeling is intense, rapid dehydration is possible (diminution of the background).

❑ Protocol a. Reagents/solutions • Dimethylformamide • 10X levamisole • Substrates — NBT — BCIP • Tris–HCl/NaCl/MgCl2 buffer; pH 9.5 b. The different steps: • Put the substrate in place 1:250 — NBT-BCIP 100 µl/ section • Incubate under visual surveil15 min lance until the desired reaction to 24 h is obtained. Darkness • Stop the reaction in distilled 5 min water. • Mount in an aqueous medium. ❷ Fast-Red

➫ (CH3)2NOCH ➫ C11H12N2S (see Appendix B6.2.1.2) ➫ Used at 1 mM

➫ See Appendix B3.7.6. ➫ For preparation, see Appendix B6.2.2.1. ➫ It is possible to accelerate the reaction by maintaining the slides at 37°C. ➫ If the degree of detection is insufficient, deposit a further 100 µl of the NBT-BCIP solution. ➫ The precipitate is soluble in alcohol. ➫ The stained reaction is expressed as a chromogen that is soluble in alcohol. ➫ C7H6N3O2 (5-chloro-2-methoxy-benzenediazonium chloride [zinc chloride]) ➫ Mw = 250.90

Figure 7.8 Fast-Red formula. 162

7.2

Immunohistochemistry

❑ Advantages • Sensitivity • Stability of the precipitate • Compatibility with multiple labels

➫ Note: Soluble in alcohol

❑ Disadvantages • The reaction sometimes takes a long time. • The precipitate is soluble in alcohol.

➫ Detection can take up to 24 h.

❑ Reagents/solutions ➫ (CH3)2NOCH ➫ Ready-to-use Fast-Red solutions are commercially available. ➫ 1 mM (see Appendix B6.2.1.2)

• Dimethylformamide • Fast-Red • 10× levamisole • Naphthol phosphate • Tris/NaCl/MgCl2 buffer, pH 9.5

➫ See Appendix B3.7.6.

❑ Protocol a. Place the filtered substrate directly on the slides extemporaneously. • Fast-Red 100 µl/ section b. Incubate under visual surveil15 min lance until the desired to 24 h reaction is obtained. rt, darkness c. Rinse with distilled water. d. Mount in an aqueous medium. 7.2.3.3

1 min

➫ For the preparation, see Appendix B6.2.1.2.

➫ The reaction is finished when the colored precipitate is red and clearly visible. It is possible to accelerate the reaction by maintaining the slides at 37°C. ➫ To stop the reaction ➫ The precipitate is soluble in alcohol. ➫ See Appendix B8.1.

Peroxidase

The peroxidase activity (oxidation of the appropriate substrate) produces an insoluble colored sub– stance (precipitate), which materializes the reaction. The chromogens (substrates) appropriate to peroxidase are:

➫ The electron donor is hydrogen peroxide.

• 3′-Diaminobenzidine tetrachloride (DAB) • 3-Amino-9-ethylcarbazole (AEC) ❶ DAB

NH2

H 2N

H 2N

NH2

➫ (3′-Diaminobenzidine tetrachloride) ➫ The substrate is oxidized in the presence of peroxidase, and produces a signal that is expressed as a yellow-brown precipitate.

Figure 7.9 The formula of DAB.

163

Revelation ❑ Advantages • • • • •

➫ Can be intensified by nickel salts ➫ Embedding in resin that is stable over time ➫ Less background noise

Brown precipitate Precipitate insoluble in alcohols Counterstaining possible Very intense reaction Double-labeling possible

➫ To be carried out as a second reaction

❑ Disadvantages • Dangerous in powder form • Possibility of background noise • Requires hydrogen peroxide of good quality • Dangerous waste

➫ Can be attenuated by preincubation with DAB, without hydrogen peroxide ➫ Hydrogen peroxide stable for only a few weeks ➫ Can be broken down by sodium hypochlorite

❑ Reagents/solutions • Diaminobenzidine tetrahydrochloride • 30% hydrogen peroxide • Tris–HCl/NaCl/MgCl2 buffer, pH 7.6

➫ C12H14N14·HCl (DAB) ➫ 110 volumes ➫ See Appendix B3.7.6.

❑ Protocol a. Place the substrate.

100 µl/ section

b. Incubate under visual surveil3–10 min lance until the desired reaction rt is obtained. c. Stop the reaction by dipping the slides in distilled water. d. Counterstain. e. Embed in resin. ❷ 3-Amino-9-ethylcarbazole (AEC)

➫ For the preparation, see Appendix B6.2.2.2. ➫ Optional. Add an enzyme-blocking agent (endogenous peroxidases, see Appendix B6.2.1.3) before the use of peroxidase conjugates. ➫ Color is brown. ➫ An overlong detection step will cause a generalized coloring of the tissue. ➫ Counterstaining is possible. ➫ The precipitate is insoluble in alcohol. ➫ The reaction is expressed as a red precipitate.

NH2

➫ C14H14N2 ➫ Mw = 210.30 N

CH2CH3

164

Figure 7.10 The formula of AEC.

7.3

Autoradiography

❑ Advantages • Red, clearly visible precipitate • Double labeling • Possibility of counterstaining ❑ Disadvantage ➫ The solvent is harmful if inhaled.

• Toxic ❑ Reagents/solutions • • • •

100 mM acetate buffer, pH 5.2 AEC (3-amino-9-ethyl carbazole) Dimethylformamide 30% hydrogen peroxide

➫ Soluble in dimethylformamide ➫ (CH3)2NOCH ➫ 110 volumes

❑ Protocol a. Place the substrate. 100 µl/section b. Incubate under visual surveil10 min lance until the desired reaction Darkness is obtained. c. Stop the reaction with distilled water. d. Embed in an aqueous medium. ➲ Following step • Observation

➫ Oxidized AEC forms a pink-red precipitate; reaction to be checked by microscope. ➫ The precipitate is soluble in alcohol (see Appendix B8.1). ➫ See Chapter 11.

7.3 AUTORADIOGRAPHY The lack of precision of the signal, the risk of contamination of the equipment, and the indispensable precautions involved in the use of radioactive isotopes mean that autoradiography is seldom used in in situ PCR or RT/PCR. A radioactive hybrid emits radiation, which is conventionally recorded by:

➫ Nonetheless, this is an approach that can be used to quantify amplification by measuring the levels of gray in an autoradiogram.

• Autoradiography, i.e., by a photographic emulsion that visualizes it, or

➫ For macroautoradiography, see Section 7.3.3.

• Phosphoimagery.

➫ For microautoradiography, see Section 7.3.4. ➫ This provides direct quantification but lower resolution.

7.3.1 Principles of Autoradiography The purpose of autoradiography is to visualize radiation or particles emitted by radioactive isotopes through their materialization as grains of metallic silver. 165

Revelation ① Slide ② Emission of radioactive particles

3

③ Detection system

2 1

Figure 7.11 Principle of autoradiography.

The isotopes most commonly used in this 35 33 method are S and P.

➫ Only these isotopes, along with β radiation, are recorded by the emulsion.

7.3.2 Characteristics of Emulsions 7.3.2.1

7.3.2.2

Radiation

Photographic emulsions

These are composed of silver bromide diluted in gelatin. They either take the form of a liquid in a gel (emulsion) or a solid (film). They are characterized by: • The size of the grains • The thickness of the layer • The type of medium

7.3.2.3

Exposure

The sample, covered by a film or a thin layer of photographic emulsion, is stored in darkness for a period of between a few hours and several months. Exposure time depends on the radioisotope, the specific activity of the probe, and the abundance of the hybrids (and thus the number of sequences looked for in the cell). 7.3.2.4

➫ The intensity of the autoradiographic signal depends, first and foremost, on the relationship between the exposure time and the half-life of the isotope. ➫ The use of autoradiographic film, like that of a phosphoimager, makes it possible to determine the exposure time without damaging the sections.

Development

The detection process changes the latent images (see Figure 7.11) resulting from the activation of the silver salts into visible metallic silver due to the radiation. − − Br + radiation → Br + e + − Ag + e → Ag

166

➫ Crucial for resolution ➫ Modifies the sensitivity and the resolution ➫ Distinction between macroautoradiography and microautoradiography

➫ Two chemical steps: • Development, with the changing of the silver salts into metallic silver by the action of radiation. • Fixation, i.e., the dissolution of the remaining salts of silver bromide.

7.3 7.3.2.5

Efficiency

The efficiency of autoradiography has not been improved for some years; it remains an extremely sensitive method.

An efficiency of the order of 15% is generally taken to be acceptable. Such a low level is explained by the following facts: • The radiation from the radioactive isotope is emitted in three dimensions, whereas the emulsion is present only on one side of the section. • The emulsion is not 100% efficient. • The detection system is not perfect. • Background is present. 35

The isotopes used ( S and degree of efficiency. 7.3.2.6

33

➫ This parameter is the result of a statistical 35 33 analysis. The isotopes used (i.e., S and P) give similar levels of resolution.

Artifacts

Artifactual labeling (background) can have a number of potential origins: • The age of the emulsion • Irregularities in the sections (striations, fissures, etc.) • Variations in the thickness of the layer of emulsion Traces of γ or α radiation The presence of chemicals Excessive prolongation of the detection step Accidental causes

7.3.2.8

➫ This is due to innumerable factors inherent in the technique.

Resolution

• The energy of the radiation • The size of the grains of emulsion • The thickness of the layer of emulsion

• • • •

➫ 15% efficiency means that it takes six disintegrations of the radioisotope to produce 1 grain of silver.

P) give the same

The distance between the source of the radiation and the silver grains depends on:

7.3.2.7

Autoradiography

➫ Some observed grains correspond to nonspecific labeling, which therefore has to be quantified and subjected to a statistical analysis. ➫ This can give rise to shadows on the sections. ➫ These can cause variations in the thickness of the emulsion layer, and thus accumulations of grains. ➫ These can occur during the dipping or drying steps, and can also result from accumulations of grains. ➫ External radiation ➫ Chemography ➫ The detection of latent pseudo-images ➫ An example is inappropriate light.

Quantification

The radioactivity is quantified by measuring: • The optical density of macroautoradiographs, or • The number of silver grains per unit area of microautoradiographs

➫ ➫ ➫ ➫

At the tissue level A rapid, standardized method At the cell level A difficult method

167

Revelation

7.3.3 Macro-Autoradiography 7.3.3.1

Overview

Macro-autoradiography is used to visualize tissue or organs by apposition of a film within the totality of a section.

➫ The film is industrially manufactured, which guarantees homogeneity and reproducibility, and thus the possibility of making comparisons between signals.

① Radiation The hybrid contains radioactive isotopes (), which emit radiation characteristic of the element in question. Autoradiography records these emissions by means of a photographic emulsion. A = film support B = photographic emulsion C = tissue or cells D = slide

② Exposure The emulsion placed in contact with the hybrids records the radiation emitted in the course of the exposure, in the form of latent images (•).

③ Development These images are turned into visible grains of silver in the course of the photographic development process. Numerous parameters are involved in the interpretation of the results.

Figure 7.12 Macro-autoradiography.

168

7.3 7.3.3.2

Autoradiography

Autoradiographic film

This varies according to: • The size of the grains • The thickness of the film • The sensitivity There are different types of autoradiographic film, each with its particular characteristics.

➫ Resolution ➫ Handling difficulties ➫ Proportional to the cost ➫ The choice of film depends on the isotope used, the density of the hybrid, and the required sensitivity.

❶ Single-coated film ❑ Advantages • Good resolution • Quantification possible

➫ Diminution of the dispersion of the radiation. ➫ The radioactivity can be quantified by measuring the optical density of the autoradiograph.

❑ Disadvantage • Generally expensive ❷ Double-coated film ❑ Advantages • Higher sensitivity • Rapid response • Possible comparison of several reactions • Quantification • Lower cost

➫ Shorter exposure time than for singlecoated film ➫ To the test of evaluation of the signal (i.e., the visualization of a possible signal) ➫ Relative quantitative analysis ➫ Measurement of the homogeneous signal density, using single-coated film ➫ Which means that it can be used as a control for exposure time

❑ Disadvantages • Macroscopic localization, medium degree of resolution • Artifacts

7.3.3.3 7.3.3.3.1

➫ Larger emission cone

Protocol EQUIPMENT/SOLUTIONS

❶ Equipment • Cassette the right size for the film • Tray the right size for the film • Autoradiographic film

➫ Regular checks should be carried out to make sure that the box is light-tight. ➫ No sterility precautions are necessary. ➫ The size of the film depends on the number of slides to be exposed.

169

Revelation ➫ Hyperfilm H, Bio Max MR, etc. ➫ X AR 500, etc. ➫ The manufacturer’s instructions for the use of the film and developer must be respected if the signal is to be optimized. ➫ Sodium thiosulfate is the basis of all fixatives. 3

— Single-coated film — Double-coated film • Developer

• Fixative ❷ Solutions • Developer

➫ Dilute according to the manufacturer’s instructions. ➫ Use 30% sodium thiosulfate, or a reagent diluted according to the manufacturer’s instructions. ➫ The characteristics of the fixative are less crucial than those of the developer.

• Fixative

˙ 7.3.3.3.2

➫ All the following steps must be carried out in a darkroom, with a safe light.

THE DIFFERENT STEPS

a. Place the completely dry sections in the autoradiographic box, attaching them to the bottom of the box. b. Place the autoradiographic film. c. Expose. • For double-coated film 24–48 h • For single-coated film

Several days

d. Store the boxes at room temperature. e. Detect. • Develop 4 min 17°°C • Rinse in running water 1 min f. Fix. • Fixative 5 min g. Rinse. • Running water • Distilled water h. Dry. i. Evaluate the signal. j. Quantify the signal.

170

30 min 1 min rt, or 37°°C

➫ Any trace of humidity will cause an autoradiographic chemoreaction, and thus background noise. ➫ Double-coated film can serve as a test for the exposure time. ➫ This is very sensitive; used for quantification purposes. ➫ Exposure time is to be determined. ➫ Following the manufacturer’s instructions ➫ Previously cooled ➫ If this duration is exceeded, the sections may be damaged. The minimum is 1 min. ➫ To avoid any trace of lime ➫ Temperature <50°C, so as not to damage the film ➫ Presence of a standard range often useful in determining the state of the film ➫ Densitometry

7.3

Autoradiography

7.3.4 Micro-Autoradiography 7.3.4.1

Overview ① Photographic emulsion Micro-autoradiography is used to visualize the silver grains individually, and to match them to cellular structures. The photographic emulsion is applied directly to the cells, in liquid form. E = photographic emulsion C = cells S = slide ② Exposure This produces latent images of the radiation emitted during exposure.

③ Detection Transformation of the latent images into visible grains of metallic silver in the course of the photographic development process. Numerous parameters are involved in the interpretation of the results. Figure 7.13 Micro-autoradiography. 7.3.4.2

Types of emulsion

The choice of nuclear emulsion is determined by the desired sensitivity. Size of the AgBr grains: • Ilford K5 • Amersham LM1 • Kodak NTB2 7.3.4.3

➫ The size of the silver bromide grains differs between emulsions.

0.2 µm 0.25 µm 0.26 µm

➫ High sensitivity ➫ Average sensitivity ➫ Average sensitivity

Advantages/disadvantages

❑ Advantages • Cellular resolution

➫ According to the isotope used, it is possible to identify different compartments in the cell. This is not the case with macroautoradiography. 171

Revelation • Quantification on a single section

➫ The thickness of the layer of emulsion must be controlled. ➫ This is done by counting the silver grains on a section.

❑ Disadvantages • The thickness of the section is difficult to control.

• Contamination is proportional to the number of slides dipped. • There are difficulties with repeatability. • Quantification is difficult between sections.

7.3.4.4 7.3.4.4.1

Protocol EQUIPMENT/REAGENTS/SOLUTIONS

❶ Equipment • Darkroom

• • • • • • •

Autoradiographic boxes Beaker/Joly tube/emulsion tube Black tape Clean histological slides Filter paper Safe light Slide-holder

• Staining trays • Water bath with the thermostat set to 43°C ❷ Reagents • Alcohol 100° • Desiccant • Nonaqueous embedding medium • Nuclear emulsion • Xylene ❸ Solutions • D 19 developer solution • Distilled water • 30% sodium thiosulfate • Stains: 172

➫ Without any particular precaution being taken, and depending on the state of the section, the surface of the emulsion is generally smooth. ➫ It also varies according to the isotope used 33 35 ( P > S ). ➫ There are variations in the thickness of the layer of emulsion from one section to another. ➫ This is essentially due to differences in the thickness and homogeneity of the layer of emulsion. Only relative values can be compared.

➫ All the following steps are carried out in a darkroom, with a safe light placed 1.50 m above the working surface, and humidity of 20 to 40%. ➫ Clean and light-tight ➫ To dilute the emulsion ➫ Light-tightness of the storage boxes ➫ Bubble test; storage of the desiccant ➫ For wiping the backs of the slides ➫ Autoradiographic room ➫ Always the same one, so that the slides are always at the same angle ➫ Nonsterile ➫ To liquefy the autoradiographic emulsion ➫ ➫ ➫ ➫ ➫

Ordinary Silica gel wrapped in filter paper See Appendix B8.2. From Amersham, Ilford, or Kodak Or another solvent of the embedding resin

➫ See Appendix B6.1.1. ➫ Nonsterile ➫ See Appendix B6.1.2.

7.3 — Methyl green, sodium acetate buffer; acetic acid, pH 4.2 — 0.02% Toluidine blue in water

7.3.4.4.2 THE DIFFERENT STEPS After dehydration and drying, the sections are dipped in the emulsion. ❶ Dilution of the emulsion a. Make two marks on a beaker, or a specially designed recipient, one to correspond to the volume of water necessary for the dilution, and the other to correspond to the total volume (water + emulsion). b. Fill with water to the lower mark. c. Leave the beaker and the emulsion at 43°C for 5 to 10 min before dilution. d. Add the emulsion with a porcelain spoon, or else pour it into the beaker until it reaches the upper mark. e. Mix carefully, and shake every 20 min. ❷ Liquefaction

1h 43°°C

❸ Coating a. Dip and remove some clean slides to clean the surface of the emulsion and eliminate air bubbles. b. Slowly dip the slides vertically in the emulsion. Remove them with a slow, uniform movement, and drain the excess emulsion on the rim of the beaker for a few seconds, then on a filter paper. ❹ Drying Dry in a vertical position. 2 h–overnight rt The emulsion must be dry on the slide before storage in the autoradiographic box. ❺ Storage Store the slides in hermetic boxes containing a dehydrating agent wrapped in aluminium foil, or placed in a black bag, at 4°°C.

Autoradiography

➫ See Appendix B7.1.4 ➫ See Appendix B7.1.6 (stock solution), can be diluted in oxalic acid neutralized by ammonia

➫ The manufacturer’s recommended dilution must be adhered to. ➫ The marks must be visible under the safe light. ➫ The temperature-regulated water bath should be equipped with beaker racks. ➫ This will liquefy the emulsion. ➫ It is possible to dilute all the emulsion, and to store it in this form in darkness at 4°C. ➫ Mix slowly (to avoid producing bubbles), using a glass rod or strip. Do not use metal instruments. ➫ The emulsion must be perfectly homogeneous. ➫ Check the temperature of the emulsion, which must be constant at 43°°C. Too high a temperature will denature the emulsion and cause background. ➫ Eliminate all the bubbles. ➫ Avoid ripples, because the thickness of the film must be uniform. ➫ Leave one slide without a section, for background noise control. ➫ The vertical position ensures the homogeneity of the layer of emulsion. ➫ If the tissue is damp, this will act on the crystals of silver bromide (AgBr), resulting in background. ➫ A dehydrating agent of the silica gel type should be used to absorb moisture. ➫ Store at 4°C to limit the development of bacteria in the emulsion. 173

Revelation ❻ Exposure time

1–3 weeks

❼ Detection

a. Take the box out at least 2 h before opening it at the temperature of the laboratory. b. Place the slides in a staining tray, and transfer them successively to the following baths: • Developer (D19) 4 min 17°°C

• Distilled water 30 s ❽ Fixation Transfer the slides successively to the following baths. • 30% sodium thiosulfate 5 min • Running water

30 min

• Distilled water 1 min ➒ Counterstaining Incubate in one of the following solutions: • 0.02% Toluidine blue in water 1–10 min rt • Methyl green, sodium acetate 1–5 min buffer; acetic acid, pH 4.2 rt Rinse in distilled water 3 × 5 min ❿ Embedding a. Dehydrate. • Alcohol 70°, 90°, 100° • Xylene

174

2×1 min/bath 2 × 2 min

➫ Sets of slides with different exposure times can be developed together. It is difficult to calculate with precision, so detection should be carried out at intervals of several days, e.g., the first after 7 days, and the others at further intervals of 7 days. ➫ The detection step is carried out in conditions identical to those for the coating of the slides, with a safe light. ➫ There is a problem of condensation. The box must be dry before it is opened. ➫ The temperature can be increased if the labeling is weak. ➫ The temperature can be lowered if there is a lot of background, which causes a diminution of the signal. ➫ The developer can be diluted to reduce the intensity of the signal. ➫ Rinse rapidly.

➫ With delicate tissue, the time can be reduced to 1 min. ➫ Note: Do the rinsing at the same temperature as the detection (17 to 18°C). Avoid temperature differences, which could cause folds in the sections. ➫ Rinse abundantly. ➫ This is a cytoplasmic and nuclear stain. ➫ Although not recommended, a slight counterstain might be performed. ➫ The nucleus is purplish-red and the cytoplasm blue. ➫ It is always possible to reduce the thickness of the gelatin layer in an acid bath. ➫ With shaking

7.3 b. Embed • Place a drop of the medium on the section. • Put a coverslip in place immediately. ➲ Following steps • Observation — Dark field — Bright field — Epipolarization • Quantification

Autoradiography

➫ Use permanent medium (e.g., Permount, Eukitt, Depex). ➫ Eliminate bubbles. ➫ Without staining ➫ With staining ➫ With staining

175

Chapter 8 Electron Microscopy

Contents

CONTENTS

1 2 3

8.1 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183

8.1.1 Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183

8.1.2 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . .

183

8.1.3 Pros and Cons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

184

8.1.4 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.4.1 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.4.2 RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

184 185 185

8.2 Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

185

Pre-Embedding Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1.1 Diagram of the Different Steps . . . . . . . . . . . . . . . 8.2.1.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . Post-Embedding Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.2.1 Diagram of the Different Steps . . . . . . . . . . . . . . . 8.2.2.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 8.2.2.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . Non-Embedding Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.3.1 Diagram of the Different Steps . . . . . . . . . . . . . . . 8.2.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 8.2.3.3 Advantages/Disadvantages . . . . . . . . . . . . . . . . . . Choice of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

185 186 187 188 188 189 189 190 191 191 192 193 193

8.3 Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

195

8.2.1

8.2.2

8.2.3

8.2.4

8.3.1 8.3.2

Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sampling Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2.1 General Precautions . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2.2 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2.3 Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

195 195 195 195 196

8.4 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

196

8.4.1 8.4.2 8.4.3

Fixative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dilution Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.3.1 Cells in Suspension . . . . . . . . . . . . . . . . . . . . . . . . 8.4.3.2 Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

196 197 197 197 198

8.5 Cutting Sections on a Vibratome. . . . . . . . . . . . . . . . . . . . . . . . . . . .

199

8.5.1 8.5.2

Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cutting Vibratome Sections: Practical Details . . . . . . . . . . . 8.5.2.1 Thickness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199 199 199 179

Electron Microscopy 8.5.2.2 Speed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.2.3 Oscillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.2.4 Adjusting the Apparatus. . . . . . . . . . . . . . . . . . . . . Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.3.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.3.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Storage of Vibratome Sections . . . . . . . . . . . . . . . . . . . . . . .

200 200 200 200 200 200 201 202

8.6 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

202

8.5.3

8.5.4 8.5.5

8.6.1 8.6.2

Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.3.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.3.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deproteinization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.4.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.4.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment with DNase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.5.1 Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.5.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Postfixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

203 203 203 203 204 204 205 205 205 206 206 206 207 207

8.7 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207

8.6.3

8.6.4

8.6.5

8.6.6

8.7.1 8.7.2

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7.3.1 The Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . 8.7.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . .

207 209 209 209 209 209 209

8.8 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

211

8.7.3

8.8.1 8.8.2

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Equipment /Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.3.1 The Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . 8.8.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 8.8.3.2.1 The Hot Start . . . . . . . . . . . . . . . . . . . . 8.8.3.2.2 The Amplification Cycles. . . . . . . . . . .

211 213 213 213 213 213 214 214 215

8.9 Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

216

8.8.3

8.9.1 8.9.2

180

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

216 217 217 217

Contents 8.9.3

Protocol for Thick Sections . . . . . . . . . . . . . . . . . . . . . . . . . 8.9.3.1 The Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . 8.9.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . .

218 218 218

Immunocytochemical Detection on Thick Sections. . . . . . . . . . . .

220

8.10.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10.3.1 Direct Reaction . . . . . . . . . . . . . . . . . . . . . . . . 8.10.3.2 Indirect Reaction . . . . . . . . . . . . . . . . . . . . . . . 8.10.3.3 Epoxy Resin Embedding . . . . . . . . . . . . . . . . .

220 221 221 221 222 222 223 224

Hydrophilic Resin Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . .

225

8.11.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.3 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.4 Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

225 225 225 226 226 226

Hybridization on Ultrathin Sections. . . . . . . . . . . . . . . . . . . . . . . .

227

8.12.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12.3.1 The Reaction Medium . . . . . . . . . . . . . . . . . . . 8.12.3.2 The Different Steps . . . . . . . . . . . . . . . . . . . . .

227 228 228 228 228 228 228

Immunocytological Detection on Ultrathin Sections . . . . . . . . . . .

229

8.13.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.13. 3 The Different Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

229 230 230 230 230

8.14 Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

231

8.14.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.2 Equipment/Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.2.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.2.2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.14.3.1 Epoxy-Resin-Embedded Tissue Sections . . . . 8.14.3.2 Hydrophilic Resin-Embedded Tissue Sections . . . . . . . . . . . . . . . . . . . . . . . . 8.14.4 Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

231 231 231 231 231 231

Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

232

8.10

8.11

8.12

8.13

8.15

232 232

181

8.1

Overview

8.1 OVERVIEW 8.1.1 Objective The objective is to detect nucleic acids that are very weakly expressed, at a subcellular level of resolution, by identifying the cytological characteristics of the cells in which they are present.

➫ This method combines the advantages of in situ PCR/RT-PCR and those of electron microscopy. ➫ It is necessary, to begin with, to make sure that the nucleic acid being looked for is not detectable simply by in situ hybridization, using electron microscopy.

8.1.2 Diagram of the Different Steps

183

Electron Microscopy

8.1.3

Pros and Cons

Visualization at the electron-microscopic level has undeniable advantages: • Visualization of cell architecture and the different organelles

• Subcellular localization of the target nucleic acid

➫ Resolution is improved by three orders of magnitude, from the µm level to the nm level. ➫ The identification of cells in heterogeneous tissue ➫ The characterization of physiological and/or pathological states ➫ The identification of the cell compartment containing the target ➫ The study of intracell movements

But there are also drawbacks, namely: • Those inherent in in situ RT-PCR methods

• Drawbacks linked to ultrastructural analysis: — Logistics — Methodology

— Confirmation of the results

➫ As described in Chapters 1 through 7, these methods involve: • Preserving target nucleic acids in situ • Making target nucleic acids accessible to the tools (primers, probes, enzymes, etc.), • Maintaining the amplified product in situ • Visualizing the signal • Checking the signal ➫ An electron microscope, an ultramicrotome, and a suitable environment are indispensable. ➫ This requires • Optimal preservation of cell structure • Ultrathin sections a few tenths of a nm thick • Compatibility of the observation method with the contrast and the signal ➫ The more finely tuned and sophisticated the technique, the more indispensable the checking procedures (see Chapter 9).

8.1.4 Applications This ultrastructural approach applies equally to the detection of: • DNA • RNA as well as to the demonstration of their absence.

184

➫ The ultrastructural in situ PCR method ➫ The ultrastructural in situ RT-PCR method ➫ Absence of contamination or transmission of a nucleic acid support

8.2 8.1.4.1

DNA

The target DNA must be exogenous to the host cell so that the cells in which it is present can be picked out among a cell population. This DNA can come from: • Viruses • Fungi • Bacteria, yeasts • Transfection products

8.1.4.2

➫ Except for the genomic DNA of the host cells ➫ Latent contamination; incorporation into the genome ➫ Contamination, symbiosis, etc. ➫ Contamination, symbiosis, etc. ➫ Studies of kinetics, effectiveness, or sexual transmission

RNA

RNA is present in only a small number of copies, which must therefore be carefully preserved. It can have different origins: • Genomic expression • Viruses • Fungi • Bacteria • The expression of a transfection product 1 2 3

Methods

➫ Measures must be taken to prevent RNase action. ➫ The most common ➫ Or the expression of a DNA virus ➫ Or DNA expression ➫ Or DNA expression ➫ The effectiveness of transfection

8.2 METHODS

In electron microscopy there are three main methods for detecting nucleic acids and proteins, and thus three corresponding approaches to in situ PCR/RT-PCR: • A non-embedding method • A post-embedding method

• A pre-embedding method

➫ This uses frozen tissue sections or cell fractions (e.g., chromosomes). ➫ The tissue or cell is first embedded in a resin (usually hydrophilic). The amplification reaction is carried out on ultrathin sections. ➫ The amplification reaction is carried out on thick sections which are then embedded in resin (epoxy or hydrophilic) before cutting ultrathin sections.

8.2.1 Pre-Embedding Method Here, as the name indicates, PCR/RT-PCR is carried out on thick sections, which are subsequently embedded in resin to make ultrathin sections for the detection of the amplified product, and for observation.

➫ 50 to 200 µm ➫ 60 to 100 nm

185

Electron Microscopy 8.2.1.1

186

Diagram of the different steps

8.2 8.2.1.2

The different steps

❶ Fixation This step requires that particular precautions be taken (fixation by perfusion, then fixation by immersion) to obtain a tissue or cell structure that is homogeneous throughout the thickness of the sample. ❷ Thick sections These are made with a vibratome. They can be from 50 to 100 µm thick. ❸ Pretreatments The aim here is twofold: • To make the tissue permeable to all the reagents • To make the target nuclear acid accessible to the reagents ❹ PCR/RT-PCR This step is carried out on floating sections in a “PCR tube.” The reaction takes place in thick sections by incubation in the reaction medium. In theory, it is possible to carry out: • Direct amplification

• Indirect amplification In practice, the difficulty of eliminating labeled nucleotides means that the direct method is lacking in specificity, and that the indirect method is strongly recommended. ❺ Washing This prevents the diffusion of amplified products. ❻ Hybridization This is carried out on thick sections before embedding. ❼ Washing This eliminates nonspecific hybrids. ❽ Detection By an indirect immunocytological reaction: a. If it is carried out before the embedding step, the antigenic label is detected by an enzyme.

Methods

➫ The possibilities of stopping the reaction are very limited in this protocol. ➫ Paraformaldehyde is the most widely used fixative.

➫ pected.

RNase-free conditions must be res-

➫ Permeabilization ➫ Deproteinization ➫ The idea is to achieve a compromise between sensitivity and the preservation of cell structure. ➫ The tube is placed in a thermocycler, which follows a predefined program of cycles. ➫ The incorporation of labeled nucleotides during the synthesis of amplified products (see Chapter 4) ➫ The detection of amplified products by an additional hybridization step (see Chapter 4)

➫ This step is crucial. It requires a negative internal control (see Chapter 9). ➫ It has been shown, in ultastructural in situ hybridization, that the penetration of the probe in the tissue is not a limiting factor. ➫ This is a step that needs to be optimized. Its effectiveness must be checked. ➫ For the detection of a hybrid bearing an antigenic label ➫ Generally peroxidase

187

Electron Microscopy b. If it comes after the embedding step, the antigenic label is detected by a colloidal gold conjugate. ❾ Embedding • In epoxy resin if the detection step comes before the embedding step. • In hydrophilic resin if the detection step comes after the embedding step and ultrathin sections are used. ❿ Contrast and observation After a more or less intense contrast, according to the chosen protocol, the sections are observed in the classical way by transmission-electron microscope.

8.2.1.3

➫ In this case the sections are easy to make. ➫ The indirect immunocytological reaction is carried out by floating ultrathin sections on drops of the different antibodies. ➫ Diaminobenzidine (DAB), a peroxidase substrate, is made opaque to electrons by contrasting with osmium tetroxide. ➫ See Section 8.10.2.2.

Advantages/disadvantages

This currently seems like the most realistic method. ❏ Advantages • Conservation of amplified products • Good morphological preservation, which facilitates the identification of subcellular structures • Utilization of a thermocycler with tubes • Possibility of multiple labeling

➫ Within thick sections ➫ Similar to that obtained with ultrastructural in situ hybridization. It should, however, be noted that in this case the nuclear structures are definitively destroyed. ➫ The handling of floating sections in PCR tubes presents no difficulties. ➫ If the detection is carried out after the embedding step. It is a delicate operation, or even impossible in the other case.

❏ Disadvantages • The operation cannot be stopped before the embedding step. • The method takes a long time. • The embedding has to be done flat, and with great care, to facilitate the recovery of the first few sections.

➫ Storage is not possible without compromising the experimental results. ➫ It requires several days. ➫ The signal diminishes as one advances further into the section.

8.2.2 Post-Embedding Method This is a classical method in electron microscopy, where the amplification reaction is carried out on ultrathin sections of resin-embedded tissue.

188

➫ At present, only hydrophilic resins give satisfactory results.

8.2 8.2.2.1

Diagram of the different steps

8.2.2.2

The different steps

❶ Fixation The most commonly used fixative is paraformaldehyde. In standard conditions, fixation is carried out by immersion.

Methods

➫ This method can be used with most hydrophilic-resin-embedded samples intended for immunocytological studies. ➫ Fixation by perfusion also gives excellent results.

189

Electron Microscopy ❷ Embedding A hydrophilic resin is used cold after partial dehydration of the tissue. Polymerization is possible with ultraviolet radiation or heat. ❸ Making ultrathin sections Their thinness is no advantage in terms of the amplification reaction, which in this case is a surface reaction. ❹ Pretreatment Deproteinization is necessary. ➎ PCR/RT-PCR Indirect amplification is used on ultrathin sections. ❻ Washing This is not very important, and of low stringency. ❼ Detection Following hybridization with an antigenic labeled probe, an indirect immunocytological reaction is the most common. Colloidal gold is the label of choice. ❽ Contrast Neutral uranyl acetate is the most commonly used. The time required is 30 or 40 min longer for hydrophilic resins than for epoxy resins. ❾ Observation This uses transmission-electron microscope at low voltage.

8.2.2.3

➫ Examples are Lowicryl or LR White. ➫ According to the type of resin used. ➫ There is little difference between these two types of polymerization. Neither facilitates the amplification reaction. ➫ In general, around 80 to 100 nm

➫ Without this, no amplification will take place. ➫ A direct reaction is possible if all the necessary checks are carried out. ➫ Because amplification is exclusively a surface reaction in these conditions, the amplified products can be removed very easily. ➫ A direct immunocytological reaction is also possible, but it is a little less sensitive. ➫ Contrastanting substances with a high pH, such as lead citrate, should be avoided, as they denature the hybrids. ➫ See Chapter 11. ➫ When sections have been weakened by temperature variations over the course of the cycle, high voltages and currents can lead to tearing.

Advantages/disadvantages

❏ Advantages • Can be used to carry out retrospective studies of embedded samples • A high degree of morphological preservation • High resolution

➫ Provided that the tissue was obtained and fixed in suitable conditions ➫ Not greatly altered by the amplification reaction ➫ A detection method using colloidal gold particles

• Rapidity ❏ Disadvantages • The accessibility of the material to be amplified is limited to the surface of the sections. • Amplified products diffuse into the reaction medium. • For observation purposes, the sections are delicate. 190

➫ There is little amplification. ➫ These products do not remain on the surface of the section. ➫ This is mostly due to the properties of the hydrophilic resin.

8.2 • The practical side of the amplification procedure.

Methods

➫ The way the grid is kept in place depends on the experimenter’s talent for improvisation.

8.2.3 Non-Embedding Method The in situ PCR/RT-PCR reaction is carried out with non-embedded tissue sections, in general with frozen sections or cell fractions placed directly on the grid of the electron microscope. 8.2.3.1

➫ Ultrathin sections are used.

Diagram of the different steps

191

Electron Microscopy 8.2.3.2

The different steps

❶ Fixation As with the two previous methods, paraformaldehyde is the most commonly used fixative. ❷ Cryoprotection The aim here is to limit the morphological consequences of the freezing process. The cell water is replaced by a water/cryoprotectant mixture so that the freezing can take place with a minimum of ice crystal formation. ❸ Freezing The aim here is to harden soft tissue in preparation for the production of ultrathin sections. ❹ Making ultrathin sections These are made by cryoultramicrotomy. They are ~100 nm thick. ❺ Pretreatments With this non-embedding method, using ultrathin sections, the proteins associated with the nucleic acids are the only limiting factor. ❻ PCR/RT-PCR This is carried out by direct incubation of the ultrathin sections placed on an electron microscope grid, on a drop of the reaction mixture. ❼ Washing To eliminate amplified products which are not linked to the tissue section, and which have diffused, washing steps are carried out on ultrathin sections by direct contact with the washing buffer. ❽ Detection The amplified product is detected directly on the sections by an indirect immunocytological reaction. The most commonly used label is colloidal gold.

192

➫ An indispensable step ➫ The cryoprotectant should interefere neither with the nucleic acids nor with the cell or tissue structures. ➫ This is the most complex step, but also the one on which the preservation of ultrastructure depends. ➫ Utilization of a cryoultramicrotome

➫ A small amount of deproteinization is sometimes necessary. ➫ The absence of an embedding structure is, paradoxically, a limiting factor for this method. After five amplification cycles, the tissue is, if not destroyed, at least unrecognizable. ➫ This treatment is brief for the same reasons as before, and the absence of an embedding medium facilitates the accessibility of the products. On the other hand, there is a high risk of eliminating all the amplified products. ➫ It is also possible to carry out a direct immunocytological reaction. ➫ It gives a better degree of resolution than DAB.

8.2 ❾ Contrast and embedding After contrasting with aqueous uranyl acetate, ultrathin frozen tissue sections are generally embedded in a medium that will limit their desiccation. The fact is that numerous artifacts are due to the loss of the water contained in the sections during the drying process. ❿ Observation 8.2.3.3

Methods

➫ Methylcellulose is the most commonly used medium. ➫ The conservation of tissue water and soluble cellular molecules gives tissue treated by this method a particular cytological appearance (grayish cytoplasm). ➫ See Chapter 11.

Advantages/disadvantages

❏ Advantages • This is a rapid method. • The resolution of the signal is good. • It is possible to use tissue or cells from collections.

➫ Using colloidal gold particles ➫ If the samples are stored in liquid nitrogen

❏ Disadvantages • The delicate nature of ultrathin frozen tissue sections limits the number of cycles that can be carried out. • The loss of cellular integrity due to the hightemperature cycles is a major disadvantage. • There is a high risk of loss of tissue architecture. • Specific equipment is required.

➫ Resulting in a limited amount of amplification ➫ Risk of the diffusion and disappearance of amplified products during the washing step ➫ Tissue organization fundamental to cell identification ➫ A cryoultramicrotome

8.2.4 Choice of Method The choice of method depends on: • Objectives • Feasibility • Reliability of detection

➫ The expected result must reply to a question. ➫ The availability of the necessary equipment and samples. ➫ According to the nature of the samples.

The following table sets out these different factors.

193

Electron Microscopy

METHODS

CRITERION Special equipment Retrospective study of samples from collections Preparation of the sample Experimental conditions Degree of simplicity Duration Reverse transcription Amplification Number of cycles Degree of diffusion of amplified products Effectiveness Detection Direct reaction Indirect reaction Label Resolution of the signal Morphological preservation Multiple labeling Feasibility Conclusion

Pre-embedding Method

Nonembedding Method

Detection before Embedding

Detection after Embedding

vibratome

vibratome

no

cryoultramicrotome

no

no

yes

yes

long

extemporaneous

extemporaneous extemporaneous

average long on floating sections on floating sections >20

average long on floating sections on floating sections >20

high short on ultrathin sections on ultrathin sections >20

average short on ultrathin sections on ultrathin sections <5

limited

limited

high

high

high

high

low

very low

possible possible antigenic or radioactive low

possible yes

no yes

no yes

antigenic

antigenic

antigenic

good

good

good

good

good

average

poor

difficult good ++

yes good +++

yes average +

possible average ±

In conclusion, the pre-embedding method is the only one that gives satisfactory results at present. The choice between the two variants of this method depends on the required resolution of the signal and the sensitivity. 194

Postembedding Method

➫ The diffusion of amplified products into the reaction medium is the major drawback with the post- and non-embedding methods. ➫ Resin-embedding limits the detection of the amplified products to the surface of the section, but it makes multiple labeling possible.

8.3

Samples

8.3 SAMPLES 8.3.1 Origin The target nuclear acid can be present in: • Cells • Tissue • An organism

➫ A biological sample from a cell culture (suspension or monolayer) ➫ Organ or biopsy ➫ Possible

8.3.2 Sampling Conditions These conditions must be optimal if the small number of copies of nucleic acid available are to be conserved, along with the morphological characteristics of the tissue or cells being studied.

➫ Whatever the level of observation (light microscopy or electron microscopy), the experimenter must always seek a compromise between the preservation of the target and that of the cytological characteristics.

The preservation of RNA targets is essentially a question of inhibiting, and if possible, completely preventing, RNase activity.

➫ Gloves must be worn. ➫ The equipment used (flasks, tubes, surgical instruments, etc.) must be either sterilized or single use.

With DNA, DNase activity is not a real problem. 8.3.2.1

General precautions

In electron microscopy, such precautions are of the classical type: • Sterility • Rapidity • Size

• Temperature

8.3.2.2

➫ Some of these precautions also apply to electron microscopy. ➫ Close-to-sterile conditions limit the risk of contamination by RNase. ➫ The sampling must be carried out rapidly to limit the risk of partial dehydration or proteolysis. 3 ➫ Of the order of mm for non- or postembedding methods. ➫ With the pre-embedding method, it can be the same as that of samples used in light 3 microscopy (1 cm ). ➫ It should be close to 4°C to limit enzymatic activity (proteolytic, RNase, etc.).

Cells

Cells come from sterile environments, and so handling them does not require any particular precautions. It is enough to eliminate the biological or culture medium and carry out a wash in a buffer after cytocentrifugation.

➫ It could interfere with the fixative. ➫ The one that contains the fixative. 195

Electron Microscopy 8.3.2.3

Tissue ① Removal of the tissue 1

② Washing in a buffer 2 3

Fixation

➲ Following step • Fixation

③ Cutting into fragments 3 1–2 mm for postembedding methods 3 0.5–1 cm for vibratome sections used in the preembedding method

Figure 8.1 Tissue sample preparation.

➫ See Section 8.4.

8.4 FIXATION The general principles and operating conditions are almost identical to those that apply to light microscopy. The differences are confined to the choice of fixative, which is more restricted, along with that of the dilution buffer and the duration of the step.

➫ See Chapter 2.

8.4.1 Fixative There are two reticulating fixatives that are compatible with the amplification reaction: • Paraformaldehyde • Glutaraldehyde

Other fixatives are relatively incompatible either with the preservation of ultrastructure (e.g., formol) or with that of the nucleic acids (e.g., osmium tetroxide). 196

➫ See Chapter 2. ➫ Usually paraformaldehyde prepared extemporaneously is used (see Appendix B4.3). ➫ The concentration must be extremely low (<0.2%) so as not to inhibit the amplification reaction. The preservation of the structures obtained is an advantage both in terms of morphological appearance and in limiting the diffusion of amplified products. ➫ For the moment, the range of compatible fixatives is limited. No comparative studies have yet been carried out.

8.4

Fixation

8.4.2 Dilution Buffer Its most important characteristics are: • Osmolarity

• pH

Drawbacks with the method: • Sterility • Composition

➫ Saline concentration is the factor that has most influence on the aqueous equilibrium, and thus the cell volume. ➫ This must always be close to neutrality so as not to disturb the equilibrium of the intracellular enzymes. ➫ The intrusion of RNase must absolutely be avoided. ➫ One of the classical buffers should be used, without any addition, e.g., phosphate buffer or PBS (see Appendix B3.4.3).

8.4.3 Protocols 8.4.3.1

Cells in suspension and monolayer cell cultures

① Cells in suspension in the fixative ② Fixation, then centrifugation ③ Removal of the supernatant ④ Resuspension, then washing

Figure 8.2 Fixation of cells in suspension.

197

Electron Microscopy

① Fixation in the culture box ② Washing

③ Scraping the cells ④ Recovery

⑤ Centrifugation ⑥ Removal of the supernatant

Figure 8.3 Fixation of monolayer cell cultures. a. Fix in a culture box. b. Wash in a buffer.

15 min 4°°C 3 × 5 min

c. Scrape the cells. d. Recover the cells in Eppendorf tubes. e. Centrifuge. <1000 g 8.4.3.2

➫ This is a cell monolayer a few µm thick. ➫ It is better to increase the number of washes than their duration. ➫ Scrape as delicately as possible. ➫ Centrifuge to remove the supernatant.

Tissue

1

2

① Immersion of the samples in the fixative ② Washing in a buffer

Figure 8.4 Fixation of tissue fragments by immersion. a. Immerse the tissue fragments in the fixative. b. Wash in an excess of buffer.

198

60–90 min 4°°C 3 × 30 min 4°°C

➫ The length of time will vary according to the size of the samples. ➫ Use the same buffer as for the fixation.

8.5 ➲ Following steps • Production of vibratome sections • Pretreatments

Cutting Sections on a Vibratome

➫ For tissue ➫ For cells

8.5 CUTTING SECTIONS ON A VIBRATOME 8.5.1 Equipment The vibratome is a vibrating microtome that is used to make thick sections (50 to 200 µm) of fixed tissue. The sections are recovered in PBS.

➫ So-called “floating” sections ① Knife holder ② Tray containing the buffer ③ The sample, attached to the nonorientable support block ④ Control panel ⑤ Magnifying glass support ⑥ Magnifying glass

Figure 8.5 Vibratome or vibrating microtome.

8.5.2 Cutting Vibratome Sections: Practical Details The sample is attached to a holder in the tray containing the buffer. The blade vibrates, cutting the tissue as it moves forward. The quality of the section depends on three parameters: • Thickness

➫ This is determined by the vertical movement of the vibrating blade.

• The speed at which the vibrating blade moves forward • The oscillation or vibration of the blade

8.5.2.1 Thickness Expressed in micrometers, it must be ≥20 µm and ≤200 µm.

➫ The structure and hardness of the tissue limit the possibilities. ➫ There are the limiting values for the use of this method. 199

Electron Microscopy 8.5.2.2

Speed

The speed at which the blade moves through the tissue will depend on the hardness and heterogeneity of the tissue.

8.5.2.3

Oscillation

This is the vibrating movement of the blade that makes the section.

8.5.2.4

➫ It will be high if the tissue is hard. ➫ It will be low if the tissue is soft and/or heterogeneous.

➫ The frequency of vibration can be chosen to suit the particular type of tissue.

Adjusting the apparatus

The settings of the apparatus take account of three characteristics of the sample: size, hardness, and homogeneity. • Size The thickness of the section rises with the size of the sample. • Hardness The harder the sample, the higher the risk that a thick section will break. Thickness will therefore be inversely proportional to hardness. • Homogeneity Slow oscillations reduce tissue heterogeneity, and give sections of better quality.

➫ Thickness:  ➫ Speed:  ➫ Oscillation:  ➫ Thickness:  ➫ Speed:  ➫ Oscillation:  ➫ Thickness:  ➫ Speed:  ➫ Oscillation: 

8.5.3 Equipment/Solutions 8.5.3.1

Equipment

• Freezer • Cyanolite • Tool for recovering the sections • Eppendorf tubes 8.5.3.2

Solutions

• Cryoprotective agents — 10% dimethylsulfoxide (DMSO) in 100 mM phosphate buffer

200

➫ −20°C, −80°C ➫ A waterproof glue ➫ Sterile (e.g., a mounted needle or a stoppered Pasteur pipette) ➫ Sterile

➫ A good cryoprotectant, but it is also an organic solvent, which may damage membranes

8.5 — 10% glycerol in 100 mM phosphate buffer — 30% saccharose in 100 mM phosphate buffer • 70° ethanol • 100 mM phosphate buffer

Cutting Sections on a Vibratome

➫ Toxic, but highly penetrative ➫ Chemically the most neutral cryprotector ➫ See Appendix B3.4

8.5.4 Protocol a. Pay attention to the positioning of the sample.

b. Adhere the sample (already fixed) to the support block. c. Fill the tray with the buffer. d. Position the blade at such a height that it grazes the sample. e. Make a number of passes with the knife to obtain a clean cutting surface. f. Choose the thickness of the 50–100 µ m sections. g. Float the sections in the buffer. h. After cutting, recover the sections with a sterile tool and place in Eppendorf tubes containing a buffer solution.

➫ The support block is not orientable, which means that the position of the sample is definitive. ➫ Use a waterproof glue (e.g., Cyanolite). ➫ Generally a phosphate buffer (see Appendix B3.4) ➫ This also allows the adjustment of the vibratome (speed and oscillation) to be checked. ➫ If a section remains attached to the sample, detach it with a sterile tool (e.g., a punch). ➫ They should be processed immediately. Storage is to be considered as a second-best option.

Figure 8.6 Thick vibratome section after fixation. ➲ Following steps • Storage • Pretreatments

➫ See Section 8.5.5. ➫ See Section 8.6.

201

Electron Microscopy

8.5.5 Storage of Vibratome Sections The only way the procedure can be interrupted is to freeze the sections immediately after they have been cut. ❑ Freezing protocol a. Divide up the required number of sections among Eppendorf tubes containing the cryoprotection agent. −20°°C −80°°C −196°°C

b. Freeze.

−20°°C A few days −80°°C A few months −196°°C Without limit

c. Store.

➲ Following step • Pretreatments

8.6 PRETREATMENTS Reverse transcription and amplification reactions are difficult to carry out on ultrathin sections, which means that thick sections and isolated cells in suspension are the only types of material that can be used in PCR/RT-PCR techniques with electron microscopy. After the fixation of the sample and the cutting of the sections, the tissue must be prepared in such a way as to render the target nucleic acid accessible to the different reagents for the amplification steps.

202

➫ The main disadvantage of the preembedding method is the length of time needed to carry it out. ➫ 30% sucrose in phosphate buffer is the most widely used. ➫ 10% glycerol or 10% DMSO in phosphate buffer are alternatives. ➫ Very slow freezing ➫ Slow freezing ➫ Rapid freezing ➫ Whatever the mode of freezing, the tissue structure will be affected. But the amount of damage will be inversely proportional to the speed of freezing. ➫ The sections are in a liquid medium, so the risk of desiccation is low. ➫ It is best to store sections in the cryoprotection liquid. ➫ The lower the storage temperature, the longer it takes for recrystallization to occur. ➫ See Section 8.6.

➫ For thick sections of the preembedding method (non-embedded tissue)

➫ Essentially by permeabilization and/or deproteinization

8.6

Pretreatments

8.6.1 Diagram of the Different Steps

8.6.2 Equipment/Solutions 8.6.2.1

Equipment

• Water bath (37°C) • Shaker 8.6.2.2

Solutions

• 9‰ NaCl • 70°, 95°, and 100° ethanol • 10 N sodium hydroxide • Buffers — 10X phosphate buffer, pH 7.4 — 20X SSC (standard saline citrate) buffer, pH 7.0 — Tris–HCl/CaCl2 buffer, pH 7.6

➫ Or incubator ➫ Slow ➫ The sterility of the solutions is crucial to the success of the following steps (see Appendix A1.1). ➫ See Appendix B2.19. ➫ If a precipitate appears, discard (see Appendix B2.20). ➫ Store for 1 month at room temperature after sterilization (see Appendix B3.4.1). ➫ Store at 4°C or room temperature (see Appendix B3.5). ➫ Store at 4°C or room temperature (see Appendix B3.7.2).

203

Electron Microscopy • Detergents — 0.2% digitonin in phosphate buffer — 0.2% saponin in phosphate buffer — 0.2% sarcosyl in phosphate buffer — 0.05% Triton X-100 in phosphate buffer • Paraformaldehyde, 100 mM 4% phosphate buffer, pH 7.4 • Proteases — 1 mg/ml proteinase K, Tris (20 mM)/ CaCl2 (2 mM) buffer, pH 7.6 — 1 mg/ml pronase, 100 mM phosphate buffer, pH 7.4 • Sterile water

➫ ➫ ➫ ➫ ➫

See Appendix See Appendix See Appendix See Appendix See Appendix

B3.4. B3.4. B3.4. B3.4. B4.3. Store at 4°C.

➫ See Appendix B2.14.1. ➫ Store at −20°C in 50 µl aliquots. ➫ See Appendix B2.14.3. ➫ Store at −20°C in 50 µl aliquots. ➫ To be used only at the time of opening the bottle. Otherwise, use DEPC water (see Appendix B1.2).

8.6.3 Permeabilization 8.6.3.1

Overview

The different reagents must be allowed access to the target nucleic acid by partially destroying the cell membranes (see Figure 8.7).

➫ This step requires a great deal of care, in that it involves a loss of cell integrity.

Figure 8.7 Permeabilized thick vibratome section. The cell membranes are made up of phospholipids, which can be solubilized by reagents such as: • Alcohol • Detergents • Enzyme ❑ Advantages • A high level of sensitivity • Partial deproteinization

204

➫ Ethanol ➫ Digitonin, saponin, or sarcosyl, Triton X-100 ➫ Lipase ➫ This produces an increase in the penetration of the different reagents. ➫ This means that the deproteinization step can be curtailed.

8.6 ❑ Disadvantages • Ultrastructural damage • An increase in the diffusion of amplified products

8.6.3.2

Pretreatments

➫ There is some loss of cellular material. ➫ The destruction of structures, and membranes in particular, favors the diffusion of amplified products. It is often recommended that after the permeabilization step the structures should be restabilized by postfixation.

Protocol

a. Incubate the sections with one of the following: • 0.05% Triton X-100 in 30 min phosphate buffer • 0.2% saponin in phosphate 30 min buffer • 0.2% sarcosyl in phosphate 30 min buffer • 0.2% digitonin in phosphate 30 min buffer b. Rinse the sections in the same buffer: • Phosphate buffer 3 × 10 min ➲ Following steps • Deproteinization • Reverse transcription • Amplification

➫ This must be tested for each new batch.

➫ Do not allow to dry out. ➫ See Section 8.6.4. ➫ See Section 8.7. ➫ See Section 8.8.

8.6.4 Deproteinization 8.6.4.1

Overview

Eliminate the proteins associated with the nucleic acids to make the latter accessible to the reagents.

➫ This is a step that needs to be checked.

Figure 8.8 Deproteinized thick vibratome section.

205

Electron Microscopy This step can be carried out using: • Enzymes

• Detergents 8.6.4.2

Protocol

a. Incubate the sections successively in: • 20 mM Tris–HCl/2 mM 15 min CaCl2 buffer, pH 7.6 rt • Proteinase K in 1–5 µ g/ml 1h Tris–HCl/CaCl2 buffer rt or 15 min 37°°C b. Rinse: • 100 mM phosphate buffer, 2 × 15 min pH 7.4

8.6.5 Treatment with DNase 8.6.5.1

➫ Specific and rapid ➫ Normally proteinase K, although other enzymes such as pronase or pepsin can also be used (see Chapter 2) ➫ Simultaneous permeabilization; but it is difficult to check the two reactions (see Chapter 2)

➫ Room temperature ➫ Incubation concentrations and times depend on the delicacy of the tissue. They must be checked for all new experiments. ➫ The choice of buffer affects the activity of proteinase K (e.g., the absence of CaCl2 lowers its activity: e.g., the use of a Tris–HCl/EDTA buffer). ➫ Here, the aim is to prevent proteinase K action, and to eliminate NH2 groups from the Tris–HCl/CaCl2 buffer. ➫ Optional

Principles

Destroys the nucleic acids present in the tissue sections by DNase I: • This prevents the formation of unwanted hybrids; • Control sections by destroying target nucleic acids.

➫ Permits selection of RNA

➫ Negative control of target nucleic acid

Figure 8.9 Thick vibratome section pretreated by DNase.

206

8.7 8.6.5.2

Reverse Transcription

Protocol

The enzyme (DNase) must be of excellent quality and not be contaminated by any other enzyme. a. Incubate the sections alternatively with: • DNase diluted in 1 mg/ml Tris–HCl/NaCl buffer 30 min 37°°C b. Wash freely in Tris– HCl/NaCl buffer.

5 × 5 min at rt

➫ This is also important for the destruction of nucleic acid targets for controls.

➫ The concentration must be tested. ➫ The duration may be increased or decreased. ➫ It is possible to work at room temperature. ➫ Or phosphate buffer (see Appendix B3.4). ➫ Indispensable ➫ The enzymes cause breaks in nucleic acids. Washing is necessary to remove all nucleic acid fragments, which may hybridize with the probe.

c. Do not let dry.

8.6.6 Postfixation a. Postfix: • 4% PF in 100 mM phosphate buffer, pH 7.4 b. Rinse the sections: • Phosphate buffer

5 min 3 × 5 min

➲ Following steps • Reverse transcription • PCR

➫ Optional step ➫ See Appendix B4.3. ➫ To eliminate the enzyme ➫ Eliminates any trace of fixative ➫ Do not allow to dry out. ➫ See Section 8.7. ➫ See Section 8.8.

8.7 REVERSE TRANSCRIPTION 8.7.1 Overview Reverse transcription turns the RNA of interest into cDNA, which is the only type of nucleic acid that can be amplified by PCR. The tools (primers, enzymes, nucleotides, etc.) are identical to those used in light microscopy.

➫ See Chapter 4. ➫ This principle is identical, whatever the level of observation: light or electron microscopy. ➫ See Chapter 4.

207

Electron Microscopy The only differences concern the protocols used with thick and floating sections.

➫ The thickness of the section will limit the penetration of the reagents, and thus the effectiveness of the reaction in depth. ➫ To overcome this disadvantage, some authors use another method consisting of placing ultrathin sections on grids which are then dipped into the reaction mixture and treated as floating sections in PCR tubes. Natif RNA:

Primer:

Enzyme:

Reverse transcription complex:

cDNA:

Figure 8.10 Reverse transcription on a thick vibratome section. 208

8.7

Reverse Transcription

➫ See Section 4.5.2.2.

It should also be noted that cells in suspension can be processed for electron-microscopic examination after the RT or PCR step.

8.7.2 Equipment/Solutions 8.7.2.1

Equipment ➫ Tube thermocycler

• Thermocycler for liquid-phase PCR • Vortex mixer 8.7.2.2 • • • • • • • • • •

Solutions

0.1 M DTT 10 µM anti-sense primer 10 mM dNTP 100 mM phosphate buffer ® 40 U/µl RNasin 5X enzyme buffer 9‰ NaCl Buffer Reverse transcriptase Sterile water

➫ ➫ ➫ ➫

See Appendix B2.7. See Section 4.3.1. See Section 4.3.2. See Appendix B3.4.

➫ ➫ ➫ ➫

See Appendix B2.19. See Appendix B3. See Section 4.3.3. See Appendix B1.1.

➫ ➫ ➫ ➫ ➫ ➫

Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/µl Final concentration: 1 µM 47.5 µl

.

8.7.3 Protocol 8.7.3.1

The reaction mixture

In a sterile microtube, prepare the reaction mixture: • • • • • •

5X RT buffer 20 µ l 0.1 M DTT 10 µ l 10 mM dNTP 5 µl 40 U/µl RNasine 2.5 µ l 10 µM anti-sense primer 10 µ l Sterile water To a total volume of 95 µ l

8.7.3.2

The different steps

Four or five thick sections are pretreated, postfixed, and placed in phosphate buffer in a PCR tube. Cells in suspension are also placed in phosphate buffer in a PCR tube.

➫ See Section 8.6.

209

Electron Microscopy

❶ Reverse transcription a. Replace the phosphate buffer by the reaction mixture, and pipette delicately to resuspend the sections. b. Add 200 U/µl reverse 5 µl transcriptase. c. Incubate in a thermocycler. 1h 37°°C 1h 42°°C d. Deactivate the enzyme. 2–15 min 94°°C ❷ Washing The sections are rinsed: • In 0.1 M phosphate 5–10 min buffer, pH 7.4 • In 9‰ NaCl 5–10 min ➲ Following step • PCR

210

➫ Final concentration: 10 U/µl ➫ The final volume is 100 µl. ➫ If reverse transcriptase is MMLV (see Section 4.3.3). ➫ If the reverse transcriptase is AMV (see Section 4.3.3). ➫ At this temperature, the enzyme is destroyed.

➫ Depending on the size of the sample ➫ Depending on the size of the sample ➫ See Section 8.8. ➫ Since the sections cannot be stored, the PCR must be carried out immediately after the RT step.

8.8

PCR

8.8 PCR 8.8.1 Overview In terms of general principles and the tools required, the amplification of a DNA sequence, whether for cells in suspension or for thick sections, is exactly the same in this case as with light microscopy. Only the subsequent steps, involving observations at the ultrastructural scale, are fundamentally different. There are two possibilities: • The indirect reaction (see Figure 8.11) • The direct reaction (see Figure 8.12)

➫ See Chapter 5.

➫ As in light microscopy, this is preferable to the direct reaction (see Chapter 4). ➫ The thickness of the sections is one possible cause of artifacts.

As for the reverse transcription step, however, it is necessary to take into account the penetration of thick sections by the reagents.

cDNA: Primers: Enzyme:

Figure 8.11 Indirect in situ PCR on a vibratome section.

211

Electron Microscopy

Amplification complex:

Amplified products:

Figure 8.11 (continued) Indirect in situ PCR on a vibratome section.

cDNA: Primers: Enzyme: Conjugated dNTP:

Figure 8.12 Direct in situ PCR on a vibratome section.

212

8.8

PCR

Labeled amplified products:

Figure 8.12 (continued) Direct in situ PCR on a vibratome section.

8.8.2 Equipment/Solutions 8.8.2.1

Equipment

• Tube thermocycler • Vortex mixer 8.8.2.2 • • • • • • • • • •

Solutions

Mixture of 10 mM dNTP 10X enzyme buffer Ethanol 100°, 95°, 70° 25 mM MgCl2 9‰ NaCl 4% paraformaldehyde 0.1 M phosphate buffer 10 µM sense and anti-sense primer  Taq or Pfu DNA polymerase Sterile water

➫ See Section 5.3.1. ➫ PCR buffer (see Chapter 5). ➫ ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B2.12. See Appendix B2.19. See Appendix B4.3.2. See Appendix B3.4.1. See Section 5.3.2. See Section 5.3.3. See Appendix B1.1.

➫ ➫ ➫ ➫ ➫ ➫

Final concentration: 1X Final concentration: 1.5 mM Final concentration: 0.2 mM Final concentration: 1 µM Final concentration: 1 µM 55 µ l

8.8.3 Protocol 8.8.3.1

The reaction mixture

In a sterile microtube placed in ice, prepare the reaction mixture: • • • • • •

10X PCR buffer 10 µ l 25 mM MgCl2 6 µl 10 mM dNTP mixture 5 µl 10 µM sense primer 10 µ l 10 µM anti-sense primer 10 µ l Sterile water To a final volume of 96 µ l

213

Electron Microscopy 8.8.3.2

The different steps

Before starting the amplification cycles, a “hot start” has to be carried out. The following washing and postfixation steps are necessary to the stabilization of the amplified products and cell structures.

8.8.3.2.1 THE HOT START  Unless one uses Pfu DNA polymerase, whose activity is minimal below 50°C, or DNA polymerase coupled to an antibody that inhibits its activity, a hot start is required to ensure the specificity of the primer matching. a. Pipette off the phosphate buffer, replace it by the reaction mixture, and carefully resuspend the sections. b. Incubate. 5 min 82°°C  c. Add 5 U/µl Taq DNA 4 µl polymerase. 214

➫ See Section 5.5.2.

➫ See Section 5.3.3.2. ➫ The risk of nonspecific hybridization is significant at low temperatures.

➫ Depending on the volume ➫ Standard temperature ➫ Final concentration: 0.2 U/µl

8.8 8.8.3.2.2 THE AMPLIFICATION CYCLES These consist of the three classical phases:

• Denaturation • Hybridization

• Extension ❶ Programming the amplification cycles: a. Three steps of PCR cycle. • Denaturation 1 min 94°°C

• Hybridization

90 s 45–60°°C

• Extension

90 s 72°°C

b. Final extension. c. Stop the reaction.

10 min 72°°C 1 min 30°°C

❷ Number of cycles This number needs to be determined empirically. It is between 10 and 25.

❸ Washing • 0.1 M phosphate buffer

X × 5 min

PCR

➫ It should, however, be noted that the amplification is carried out in a tube in a larger reaction volume than for liquid-phase PCR. This volume determines how long the different phases of the cycle need to last so that the temperatures are respected. ➫ See Section 5.3. ➫ It must be effective within all the cells. ➫ See Section 5.3. ➫ It must also be thermally compatible with the set of primers, and facilitate their diffusion. ➫ See Section 5.3. ➫ It must also last long enough to allow the enzyme and the nucleotides to diffuse. ➫ The duration has to be optimized, and the absence of a signal can be due to insufficient denaturation. ➫ This temperature is considered a reference value. ➫ Poor hybridization may result in a weak signal. ➫ The hybridization temperature must be optimized according to the characteristics of the primers. ➫ Insufficient extension may result in a weak signal. ➫ This is a reference value for most DNA polymerases. ➫ Necessary ➫ Reference value ➫ The sections can be left waiting for some time. ➫ The thermocycler can be set at 4°C if the sections are left waiting for some time. ➫ Too few cycles result in insufficient amplification, but with larger numbers there is a risk of destabilization of the structures, and loss of the amplified products, by diffusion. ➫ During systematic assays, the amplification signal decreases after 30 cycles. ➫ The aim is elimination of diffused products. ➫ The number (X) of washes, and their duration, should be high. ➫ Resuspend the sections after each wash.

215

Electron Microscopy ❹ Postfixation An important step for: • Stabilizing tissue and cell structures • Fixing the amplified products

— 4% paraformaldehyde — 0.1 M phosphate buffer — 9‰ NaCl

5–10 min 5 min 2 min

➲ Following steps • Hybridization • Embedding

➫ Observation and ultrastructural analysis require good morphological quality. ➫ The fragility of cell structures due to pretreatments (e.g., permeabilization, deproteinization), and temperature variations during the amplification cycles result in the diffusion and loss of amplified products during the detection steps. ➫ The foregoing baths are completely eliminated by pipetting, and the sections are carefully resuspended. ➫ There is no possibility of storage or of leaving the sections waiting. ➫ See Section 8.9. ➫ See Section 8.10.

8.9 HYBRIDIZATION 8.9.1 Overview With indirect in situ PCR/RT-PCR, hybridization is the means used to detect amplified products, both in electron and light microscopy. The neosynthesized DNA is double stranded, and so two specific probes, sense and anti-sense, are used. These are taken from the fragment of interest so that it alone will be detected.

➫ See Chapter 6. ➫ A single probe can be used, but this will reduce the effectiveness by half. ➫ An increase in specificity.

Denaturation of the amplified products:

216

8.9

Hybridization

Probes:

Incubation in the presence of probe

Labeled hybrids:

Formation of labeled hybrids

Figure 8.13 Hybridization of amplified product on a vibratome section.

8.9.2 Equipment/Solutions 8.9.2.1

Equipment

• Incubator • Shaker • Vortex mixer

8.9.2.2

Solutions

• Deionized formamide • 50X Denhardt’s solution • 10 mg/ml salmon sperm DNA

➫ See Appendix B2.4. ➫ See Appendix B2.5. ➫ See Appendix B2.8.

217

Electron Microscopy ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫

• Labeled anti-sense probe

• Labeled sense probe

• 10 mg/ml tRNA • 20X SSC • Sterile water

8.9.3 Protocol for Thick Sections 8.9.3.1

1.25 pmol/µl for an antigen 0.25 pmol/µl for a radioisotope See Chapter 6. 1.25 pmol/µl for an antigen 0.25 pmol/µl for a radioisotope See Chapter 6. See Appendix B2.15. See Appendix B3.5. See Appendix B1.1.

➫ For the ultrathin section protocol, see Section 8.12.

The reaction mixture

In a sterile microtube placed in ice, prepare the reaction mixture: • • • • •

20X SSC Deionized formamide 50X Denhardt’s solution 10 mg/ml tRNA 10 mg/ml salmon sperm DNA • Labeled sense probe

100 µ l 250 µ l 10 µ l 12.5 µ l 12.5 µ l 8 µl

• Labeled anti-sense probe 8 µl • Sterile water To a final volume of 500 µ l

8.9.3.2

Final concentration: 4X Final concentration: 50% Final concentration: 1X Final concentration: 250 µg/ml Final concentration: 250 µg/ml

➫ Final concentration: • 20 pmol/ml for antigens • 4 pmol/ml for radioisotope ➫ Final concentration: 20 pmol/ml ➫ 99 µ l

The different steps

The classical hybridization procedure is followed, with the same temperatures and durations. The sections are incubated either in PCR tubes (for floating sections) or on glass slides (for semifloating sections). With cells in suspension, hybridization is carried out in tubes.

218

➫ ➫ ➫ ➫ ➫

➫ The amount of handling involved is reduced, and therefore in the risk of drying out. ➫ Here, the handling of the sections is a delicate matter, but the reaction volume necessary for the incubation of sections that are well spread on slides is lower. ➫ After the hybridization step, the cell pellet is treated in the same way as a tissue sample: it is embedded and cut into sections for immunocytochemical detection.

8.9

Hybridization

Amplification

Washes

Floating sections

Semifloating section

Cell suspensions

Hybridization

Washes

❶ Hybridization a. Replace the phosphate buffer in which the sections are dipped by the reaction mixture. b. Resuspend the sections.

c. Denature.

d. Cool immediately by contact with ice. e. Incubate in the thermocycler.

5 min 96°°C 5 min Overnight

40°°C ❷ Washing a. Remove the reaction mixture by pipetting. b. Rinse: • 4X SSC • 2X SSC • 0.5X SSC

2 × 10 min 2 × 30 min 30 min

➫ Or spread the sections on slides ➫ Or, in the case of semifloating sections, cover the sections with the reaction mixture and a coverslip to avoid evaporation ➫ The amplified product is double stranded, so it has to be denatured before hybridization. ➫ On a heating block ➫ To stabilize the DNA in single-strand form ➫ The slides are incubated in a moisture chamber containing 5X SSC to maintain the same vapor saturation between the reaction mixture and the moist environment. ➫ The hybridization temperature is specific to the probes being used. It can be increased to improve specificity. ➫ If necessary, detach the coverslips with 4X SSC. ➫ Or resuspend the sections in decreasing concentrations of SSC ➫ Necessary to optimize the stringency of the washing in terms of the results (see Chapter 6) ➫ At room temperature ➫ At room temperature ➫ At room temperature 219

Electron Microscopy ➲ Following steps Either:

➫ Immediately after the RT step ➫ See Section 8.10.

• Detection using an antigenic probe and floating sections, and • Embedding; or:

➫ See Section 8.10.3.3.

• Embedding in hydrophilic resin, and • Antigenic detection on ultrathin sections.

➫ See Section 8.11.

➫ See Sections 8.12 and 8.13.

8.10 IMMUNOCYTOCHEMICAL DETECTION ON THICK SECTIONS 8.10.1 Overview The label of the hybrids present in vibratomee sections is considered an antigen. Its detection is obtained by an immunocytochemical reaction. The limitation of this detection results from the penetration of the different reagents (antibodies, labels, chromogens).

➫ See Figure 8.14.

Antibody:

Figure 8.14 Immunocytological detection on vibratome section. Two types of immunocytochemical reaction can be carried out after hybridization and washing: • Direct reaction, in which the antigen/ antibody complex is labeled.

220

➫ See Figures 7.3 and 7.4.

➫ Utilization of a primary antibody: • Immunoglobulins of polyclonal or monoclonal origin (IgG), or • Fragments of IgG: Fab or F(ab′)2, conjugated to a label

8.10 • Indirect reaction, in which the antigen/ antibody complex formed during the first step is not labeled. This is the second step, with the second conjugated antibody, which reveals the formation of the complex. The most widely used label for this detection method using floating sections is peroxidase, an enzyme that catalyzes the precipitation of chromogen. ❑ Advantage • This reaction takes place within the thickness of the section.

Immunocytochemical Detection on Thick Sections ➫ The use of a nonconjugated primary antibody, followed by a second conjugated antibody directed against the species that was used to make the first one

➫ Diaminobenzidine (DAB) is the most widely used chromogen. It oxidizes into a brown precipitate, and is made opaque to electrons by osmicated postfixation. ➫ Unlike colloidal gold, which, in spite of the use of small-sized particles (1 to 5 nm), is a surface label.

❑ Disadvantage • The precipitate is diffused, and often does not give the precision required for observation at the subcellular level.

8.10.2 Equipment/Solutions 8.10.2.1 Equipment • • • •

Horizontal slow shaker 16-well culture box Cover slide made of silicon glass Vortex mixer

8.10.2.2

Solutions

• Buffers: — blocking buffers — PBS buffer — Tris–HCl/NaCl buffer, pH 7.6 — Tris–HCl/NaCl/ovalbumin buffer, pH 7.6

• • • • •

➫ Sterile ➫ See Appendix A3.2.

— Tris–HCl/NaCl/goat serum/Triton X-100 buffer, pH 7.6. Ethanol 30°, 50°, 70°, 80°, 90°, 95°, 100° Fixative — 1% osmium tetroxide/PBS Detection solution (DAB) Hydrogen peroxide Direct reaction antibody — conjugated anti-label IgG

➫ See Appendix 6.2.1. ➫ See Appendix B3.4.3. ➫ See Appendix B3.7.5. ➫ Other agents can be added to the blocking buffer (e.g., fish gelatin, ovalbumin, nonsweetened skimmed milk). ➫ See Appendix B3.7.5. ➫ Add 1% goat serum and 0.01% Triton X-100. ➫ See Appendix B3.4.3. ➫ See Appendix B6.2.2.2. ➫ 30% H2O2 = 110 volumes. ➫ Monoclonal or polyclonal IgG, Fab fragments ➫ Conjugated = peroxidase. 221

Electron Microscopy • Indirect reaction antibody — Conjugated anti-label IgG (species X) — Conjugated anti-species X IgG • Epon • Epoxy inclusion medium — Hexahydrophtalic anhydride — Succinic dodecenyl anhydride — Nadic methyl anhydride (dicarcoxylic norborene anhydride-2,3) • 2,4,6-Tridimethylamine methyl phenol • Propylene oxide

➫ Monoclonal or polyclonal IgG, or F(ab′)2, Fab IgG fragments ➫ Peroxidase ➫ Epoxy resin ➫ See Appendix B5.2 ➫ Epikote 812 ➫ DDSA (hardener) ➫ MNA (hardener) ➫ DMP 30 (accelerator)

8.10.3 Protocol 8.10.3.1

Direct reaction

a. Incubate thick sections in a 16-well culture box: ≥200 µ l/ • Tris–HCl/NaCl buffer, pH 7.6 thick section 10 min b. Block nonspecific sites: ≥200 µ l/ • Tris–HCl/NaCl buffer; ovalbumin, thick section pH 7.6 60 min rt c. Inhibit endogenous enzymatic activity:

• Hydrogen peroxide for endogenous peroxidase

3:100 in Tris–HCl buffer 3–5 min d. Incubate in the conjugated anti-label antibody: ≥200 µ l/ • Diluted to 1:100 in Tris–HCl/ NaCl buffer, thick section pH 7.6 • Incubate.

222

1 night 4°°C

➫ The sections must never dry out. ➫ To reequilibrate the tissue, after the posthybridization washings, in a buffer that is better than SSC for immunocytochemical reactions ➫ Indispensable for limiting nonspecific reactions ➫ Optional, since endogenous peroxidase can be destroyed during the different hightemperature stages of the PCR (see Appendix B6.2.1.3) ➫ Its existence can be checked by leaving out the conjugated antibody. ➫ 30% H2O2 = 110 volumes

➫ Formation of the antigen–antibody complex. ➫ Primary antibody ➫ The dilution of the antibody is generally recommended by the manufacturer, but can be adapted to the intensity of the observed signal.

8.10 e. Rinse: • Tris–HCl/NaCl buffer, pH 7.6 f. Detect peroxidase activity: • 0.025% DAB; Tris– HCl buffer; 0.03% hydrogen peroxide; pH 7.6 g. Incubate under visual surveillance.

3 × 10 min

➫ An important step ➫ Increase the number of washings to improve the signal/background ratio

100 µl/thick section

➫ For the method of preparation, see Appendix B6.2.2.2.

3–20 min rt

➫ Color is brown. ➫ Too long a detection process leads to a fall in the signal/background ratio. ➫ Stop when the intensity and the signal/background ratio are correct. ➫ Indispensable before osmium tetroxide

h. Stop the reaction: • PBS i. Postfix vibratome sections: • 1% osmium tetroxide PBS j. Rinse: • PBS ➲ Following step • Embedding in epoxy resin 8.10.3.2

Immunocytochemical Detection on Thick Sections

5–30 min 30 min 3 × 10 min

➫ The DAB chromogen is made opaque to electrons by the reduction of osmium atoms. ➫ Rinse to eliminate the fixative. ➫ See Section 8.10.3.3.

Indirect reaction

The first steps, and the method of carrying them out, are identical to those of the direct reaction: • Blockage of nonspecific sites • Inhibition of endogenous enzymatic activity The changes begin in step d. d. Incubate the anti-label antibody (species X). • Diluted 1:100 in Tris– HCl/NaCl buffer, pH 7.6

• Incubate

≥200 µ l/ thick section

➫ Primary antibody ➫ The dilution of the antibody is generally recommended by manufacturers, but it must be optimized to improve the signal/background ratio.

Overnight 4°°C

e. Rinse • Tris–HCl/NaCl 3 × 10 min buffer, pH 7.6 f. Incubate the conjugated anti-species-X antibody.

• Incubate

➫ Formation of the antigen/antibody complex

≥200 µ l 2h rt

➫ Increase the number of washes to improve the signal/background ratio. ➫ Conjugated secondary antibody: IgG or fragments, diluted according to the manufacturer’s recommendations ➫ Dilution < that of the primary antibody ➫ Can be reduced if necessary

223

Electron Microscopy g. Rinse further. • Buffer Tris–HCl/ 3 × 10 min NaCl; pH 7.6 The steps in the direct reaction, i.e.: • Detection of peroxidase activity, • Checking the reaction, and • Postfixation of vibratome sections with osmium tetroxide are identical to those of the indirect reaction. ➲ Following steps • Embedding in epoxy resin • Embedding in hydrophilic resin

8.10.3.3

Epoxy resin embedding

❶ Dehydration Ethanol 30° Ethanol 50° Ethanol 70° Ethanol 80° Ethanol 90° Ethanol 95° Ethanol 100°

15–30 min 15–30 min 15–30 min 15–30 min 3 × 15–30 min 3 × 15–30 min 3 × 15–30 min

❷ Infiltration a. Infiltrate thick sections in solvent/resin mixtures at increasing resin concentrations: • Propylene oxide + 2:1 v/v Epon 45 min • Propylene oxide + 1:1 v/v Epon 45 min • Propylene oxide + 1:2 v/v Epon 45 min b. Replace the substitute solution with Epon resin: • First bath: Epon + DMP 30 224

➫ See Section 8.10.3.1. ➫ See Section 8.10.3.1. ➫ See Section 8.10.3.1.

➫ See Section 8.10.3.3. ➫ See Section 8.11. ➫ Only in the case of multiple labeling (i.e., the identification of a particular protein using colloidal gold after the detection of amplified products)

This protocol for embedding in Epon resin is classical.

• • • • • • •

➫ An important step ➫ Use an excess of buffer.

1–2 h rt

➫ All the other epoxy resins (e.g., araldite, spurr) can be used with the classical protocols. ➫ The sections are processed on 16-well plates. ➫ All the following steps are carried out at room temperature. ➫ With each change of bath, eliminate as much of the previous liquid as possible.

➫ The total dehydration of the samples is indispensable. ➫ Prepare the different infiltration solutions extemporaneously. ➫ Use stoppered flasks. ➫ Some types of plastic do not withstand propylene oxide. ➫ Steps are carried out at room temperature. ➫ This is a toxic mixture, to be handled under a ventilated hood.

➫ Use a freshly prepared resin. ➫ Use an unstoppered flask, to obtain the complete evaporation of the solvent. ➫ This is a toxic mixture, which must be handled under a ventilated hood.

8.11 • Second bath: Epon + DMP 30 ❸ Embedding

Overnight 4°°C

a. Spread the thick sections with a fine point: • In a mold, for flat embedding • On a coverslip made of silicon glass, taking care to eliminate as much resin as possible each time. b. Place a capsule filled with resin vertically on the section. ❹ Polymerization • Duration 48 h 60°°C ➲ Following steps • Ultramicrotomy

Hydrophilic Resin Embedding

➫ Avoid air bubbles. ➫ 300 µl of resin per cavity is used in flat embedding. ➫ This is a toxic mixture, which must be handled under a ventilated hood. ➫ With flat embedding, the sections can be arranged in any given plane. ➫ For the preparation of the coverslip, see Appendix A3.2.

➫ To facilitate removal from the molds, it is recommended that they be cooled to 4°C for 15 to 30 min. ➫ Making semithin sections ➫ Making ultrathin sections

• Observation

8.11 HYDROPHILIC RESIN EMBEDDING ➫ This step can be carried out: • After amplification, • After hybridization, or • After immunocytochemical detection

8.11.1 Overview Vibratome sections are embedded in LR White resin or another hydrophilic resin. The protocol is classical, and comprises three steps:

➫ Of the Lowicryl or Unicryl type

• Dehydration, • Substitution, and • Polymerization followed by the making of ultrathin sections, either directly observed after contrasting or on which the detection of hybrids is carried out.

➫ See Section 8.13.

8.11.2 Equipment/Solutions 8.11.2.1 Equipment • Incubator • Ultramicrotome • Ultraviolet polymerization system

➫ >60°C ➫ If possible, at low temperature 225

Electron Microscopy 8.11.2.2

Solutions

• Alcohol 50°, 70°, 95°, 100° • Hydrophilic resin — LR White — Lowicryl K4M

➫ See Appendix B5.3. ➫ See Appendix B5.3.2. ➫ This can be used instead of LR White. Its toxicity is a disadvantage, and it must be used only in a cryosubstitution apparatus of the AFS type (Automatic Freeze-substitution System). ➫ See Appendix B5.3.1.

8.11.3 The Different Steps ❶ Dehydration • Alcohol 50°, 70°, 95°, 10 min 100° per bath ❷ Substitution • Alcohol 100°–LR White 30 min (2:1 v/v) • Alcohol 100°–LR White 30 min (1:1 v/v) • Alcohol 100°–LR White 30 min (1:2 v/v) ❸ Polymerization The section, having been well spread on a very flat surface in a drop of resin, is covered with a capsule. Then it is either: • Placed in an incubator,

2 days 60°°C

• Subjected to ultraviolet radiation

2–3 days −20°°C

or

➫ Identical for other resins . ➫ Very similar for other resins

➫ Polymerization takes place only in anaerobic conditions. Oxygen prevents polymerization. ➫ Polymerization is an exergonic reaction, which favors the denaturing of hybrids and/or amplified products. It should be carried out before hybridization. ➫ This can cause breaks in amplified products. ➫ It should be carried out after detection on the sections.

8.11.4 Sections 100-nm-thick sections are classically made on an ultramicrotome.

They are collected on collodionized and carbonized nickel grids.

226

➫ After the immunocytochemical detection has been carried out, only the first sections give a high level of signal. Great care must therefore be taken in preparing them. ➫ In other cases, it is preferable to leave out the first few sections, which always have mechanical artifacts due handling. ➫ Particularly important if other processes have to be carried out on the sections, e.g., autoradiography or hybridization.

8.12 ➲ Following steps • Hybridization • Immunocytological detection

Hybridization on Ultrathin Sections

➫ See Section 8.12. ➫ See Section 8.13.

8.12 HYBRIDIZATION ON ULTRATHIN SECTIONS 8.12.1 Overview If this step in the detection of the indirect in situ PCR/RT-PCR is not carried out on thick sections, it is possible to carry it out on ultrathin sections. The principle is identical after the denaturing of the amplified products, and these are hybridized with two specific probes.

➫ See Chapter 5. ➫ The sections are embedded in hydrophilic resin after the PCR step.

Denatured hybrids: ①

① Denaturation

Probes: ②

② Incubation with the probes

Labeled hybrids: ③

③ Formation of labeled hybrids Figure 8.15 Hybridization on ultrathin section.

❑ Advantages • Resolution • Multiple labeling • Morphological preservation • Limitation of problems due to the penetration of tools (i.e., probes and antibodies) during the detection process

➫ Colloidal gold particles can be used for detection (see Section 8.13). ➫ Simultaneous detection of the different amplified products is possible. ➫ Hybridization causes little or no damage to sections. ➫ See Chapter 7.

❑ Disadvantage • Loss of sensitivity

➫ Hybridization is limited to the surface of the sections.

227

Electron Microscopy

8.12.2 Equipment/Solutions 8.12.2.1

Equipment

• Slow shaking device • Petri dish • Needle-nosed pliers

8.12.2.2 • • • • •

Solutions

50X Denhardt’s solution 10 mg/ml salmon sperm DNA Deionized formamide 0.5 N NaOH Labeled anti-sense probe

• Labeled sense probe • 10 mg/ml tRNA • 20X SSC • Sterile water

8.12.3 Protocol 8.12.3.1

➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B2.5. See Appendix B2.8. See Appendix B2.4. See Appendix B2.20. 1.25 pmol/µl See Chapter 6. 1.25 pmol/µl See Chapter 6. See Appendix B2.15. See Appendix B3.5. See Appendix B1.1.

➫ For the protocol used with thick sections, see Section 8.9.3.

The reaction medium

In a sterile microtube placed in ice, prepare the reaction mixture: • • • • • • • •

20X SSC 100 µ l Deionized formamide 150 µ l 50X Denhardt’s solution 20 µ l 10 mg/ml tRNA 12.5 µ l 10 mg/ml salmon sperm DNA 12.5 µ l Labeled sense probe 8 µl Labeled anti-sense probe 8 µl Sterile water To a final volume of 500 µ l

8.12.3.2

Final concentration: 4X Final concentration: 30% Final concentration: 2X Final concentration: 250 µg/ml Final concentration: 250 µg/ml Final concentration to 20 pmol/ml Final concentration to 20 pmol/ml 189 µ l

The different steps

The amplified products that are present at the surface of the sections are considered exogenous DNA. They are first denatured, then hybridized with two probes, each of which is complementary to one of the strands of this DNA. The excess of the probes, and nonspecific hybrids, are eliminated by washings. 228

➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫

➫ The ultrathin sections are incubated by floating the grid on the surface of a drop of hybridization buffer, and washed in the same way.

8.13 ❶ Denaturation a. Float the grids on a solution: • 0.5 N NaOH b. Wash: • SSC buffer

Immunocytological Detection on Ultrathin Sections

15 min rt 4X 3 × 5 min rt

❷ Hybridization Incubate the sections on the reaction mixture.

3h rt

❸ Washings Incubate the sections successively on drops of: • 4X SSC • 2X SSC • 0.5X SSC

2 × 10 min 2 × 30 min 30 min

➲ Following step • Immunocytological detection on ultrathin sections

➫ The amplified product is double stranded, so it has to be denatured before hybridization. ➫ NaOH must be eliminated. ➫ To avoid evaporation, this should be carried out in a moisture chamber in 5X SSC. ➫ A longer duration rarely improves the intensity of the reaction. ➫ The temperature can be increased to improve specificity. ➫ If necessary, remove the coverslips with 4X SSC. ➫ The stringency of the washings may vary in line with the results (see Chapter 6). ➫ At room temperature ➫ At room temperature ➫ At room temperature ➫ No possibility of storage ➫ See Section 8.13.

8.13 IMMUNOCYTOLOGICAL DETECTION ON ULTRATHIN SECTIONS 8.13.1 Overview The label or haptene incorporated into the hybrids formed during the hybridization step is considered as an antigen. Its detection takes place through the formation of an antigen/antibody complex in a direct or indirect immunocytological reaction, most often using colloidal gold for visualization.

➫ Identical to that of the immunocytochemical detection method used with thick sections (see Section 8.10)

Antibody:

Colloidal gold particle:

•

Figure 8.16 Immunocytological detection on an ultrathin section.

229

Electron Microscopy

8.13.2 Equipment/Solutions 8.13.2.1

Equipment

• Needle-nosed pliers • Parafilm® • Petri dish 8.13.2.2

Solutions

• An anti-species antibody conjugated to colloidal gold particles

• Anti-haptene antibodies

• Distilled water • 2.5% glutaraldehyde • Buffers — Tris–HCl/NaCl, pH 7.4 — Tris–HCl/NaCl, pH 8.2 — Tris–HCl/NaCl/ovalbumin/Tween 20, pH 8.2 — 20X SSC

➫ See Chapter 7. ➫ The size of the colloidal gold particles is generally 10 nm, but 5-nm particles can be used as a compromise between resolution and sensitivity. ➫ The most frequently used haptenes are digoxigenin, biotin, and fluorescein. ➫ See Chapter 6. ➫ RNase-free conditions are not necessary. ➫ See Appendix B1.1. ➫ See Appendix B4.2. ➫ See Appendix B3. ➫ See Appendix B3.7.5. ➫ See Appendix B3.7.5. ➫ See Appendix B3.7.5; added with 1% ovalbumin and 0.1% Tween 20. ➫ See Appendix B3.5.

8.13.3 The Different Steps ❶ Indirect detection by an antibody directed against the antigenic molecule used. a. Incubate: • Anti-haptene monoclonal antibody, 1h diluted to 1:50 in Tris–HCl/NaCl rt buffer, pH 7.4, to which a blocking agent has been added b. Rinse: • In the same buffer 2 × 5 min • In Tris–HCl/NaCl/ovalbumin/ 2 × 5 min Tween 20 buffer, pH 8.2 c. Incubate. • Anti-mouse antibody conjugated to 1h 10-nm colloidal gold particles, diluted rt to 1:50 in the same buffer as before d. Rinse: • Tris–HCl/NaCl buffer, pH 8.2 2 × 5 min • 2X SSC buffer 2 × 5 min

230

➫ According to the label used ➫ Drops of the reagent are placed on a plastic film of the Parafilm type. The grids are incubated on drops of reagent, with the section side against the liquid. ➫ For blocking buffer, see Appendix B6.2.1. ➫ A pH of 8.2 important for the stability of the antibody conjugated to colloidal gold ➫ According to the primary antibody used ➫ Anti-species antibody directed against the species of the first antibody

➫ To eliminate all trace of Tris–HCl buffer, which could interfere with the fixation step

8.14 ❷ Fixation

Staining

➫ Stabilization of the immunocytological reaction

• 2.5% glutaraldehyde in 2X SSC buffer • Washing in 2X SSC • Rapid rinsing in distilled water ➲ Following steps • Staining • Observation

5 min 2 × 5 min ➫ See Section 8.14. ➫ See Chapter 8.11.

8.14 STAINING 8.14.1 Overview Place heavy metal salts (e.g., uranium, osmium, lead) in contact with the surface of the ultrathin section to render the compounds opaque to electrons. Figure 8.17 An ultrathin section after staining.

8.14.2 Equipment/Solutions 8.14.2.1 • • • •

Equipment

Needle-nosed pliers Parafilm Petri dish Wash bottle

8.14.2.2

Solutions

• Uranyl acetate: — Aqueous 5% — Alcoholic 2% • Lead citrate • Distilled water

➫ ➫ ➫ ➫

See Appendix B7.2.1.2. See Appendix B7.2.1.1. See Appendix B7.2.2. RNase-free conditions are not necessary.

8.14.3 Protocol 8.14.3.1

Epoxy-resin-embedded tissue sections

a. Incubate the grids on a drop of: • Alcoholic uranyl acetate

2% 15–30 min

➫ The incubation time has to be controlled. 231

Electron Microscopy b. Rinse the grids in: • Distilled water

2 × 5 min

c. Incubate the grids on a drop of: • lead citrate

d. Rinse the grids in: • Distilled water

8.14.3.2

2% 2–5 min

➫ The water must be filtered through a 0.2 µm filter. ➫ The incubation time has to be controlled.

2 × 5 min Wash bottle jet

Hydrophilic resin-embedded tissue sections

a. Incubate the grids on a drop of: • Aqueous uranyl acetate

5% 30 min

➫ Good contrast is difficult to obtain on hydrophilic resin sections with aqueous uranyl acetate. The incubation time must be optimized.

2 × 5 min Wash bottle jet

➫ The water must be filtered through a 0.2 µm filter.

The grids are stored in conditions free from dust and ultraviolet radiation, pending observation.

➫ Ultraviolet radiation is not used with epoxy resin-embedded sections.

b. Rinse: • Distilled water

c. Dry the grids.

8.14.4 Storage

8.15 OBSERVATION The grids are observed by transmission electron microscopy at 60 or 80 kV.

232

➫ See Chapter 11.

Chapter 9 Controls and Problems

Contents

CONTENTS 9.1

9.2

9.3

9.4

Signal/Background Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.1 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.2 Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.3 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.4 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.5 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.6 Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.7 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sensitivity/Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.1 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2 Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.3 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.4 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.5 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.6 Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.7 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.8 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.1 Tools and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.2 Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.3 Pretreatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.4 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.5 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.6 Hybridization/Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.7 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.8 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.9 Validation by Other Techniques . . . . . . . . . . . . . . . . . . . . . . . . 9.3.10 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . False Positives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.1 Nonspecific Incorporation of Labels . . . . . . . . . . . . . . . . . . . . . 9.4.1.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.1.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.1.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.2 Repair of Cellular DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.2.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.2.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.2.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.3 Nonspecific Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.3.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.3.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.3.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.4 Nonspecific Hybridization of the Primers . . . . . . . . . . . . . . . . . 9.4.4.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . .

237 237 238 239 240 241 242 242 243 243 244 245 246 248 249 249 250 256 256 257 257 258 259 259 260 260 261 262 263 264 264 264 264 264 265 265 265 265 265 265 266 266 266

235

Controls and Problems

9.5

236

9.4.4.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.4.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.5 Amplification of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . 9.4.5.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.5.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.5.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.6 Diffusion of Amplified Products . . . . . . . . . . . . . . . . . . . . . . . . 9.4.6.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.6.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.6.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.7 External Contamination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.7.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.4.7.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.7.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.8 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . False Negatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.1 Destruction of Target Sequences . . . . . . . . . . . . . . . . . . . . . . . . 9.5.1.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.1.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.1.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.2 Problems Related to the Fixation Process . . . . . . . . . . . . . . . . . 9.5.2.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.2.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.2.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.3 Problems Related to Proteasic Digestion . . . . . . . . . . . . . . . . . 9.5.3.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.3.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.3.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.4 Reverse Transcription Problems . . . . . . . . . . . . . . . . . . . . . . . . 9.5.4.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.4.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.4.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.5 Amplification Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.5.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.5.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.5.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.6 The Stringency of the Washes . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.6.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.6.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.6.3 Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.7 Detection Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.7.1 Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . 9.5.7.2 Causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.7.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.8 Summary Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

266 266 266 267 267 267 267 268 268 268 268 268 268 268 269 269 270 270 270 270 271 271 271 271 272 272 272 272 272 273 273 273 273 273 274 274 275 275 275 275 275 275 275 276 276

9.1

The quality of the detection method depends on the optimization of each stage in the procedure, from the sampling conditions to the revelation process itself. An interpretable result is obtained by respecting the fundamental principles and modifying certain factors. The validity of this result is underpinned by the numerous controls that have to be carried out.

Signal/Background Ratio

➫ See Sections 9.1 and 9.2. ➫ See Section 9.3.

9.1 SIGNAL/BACKGROUND RATIO Result

=

Signal

=

+

Background

+

➫ The observed result comprises the signal plus the background. Only the ratio of these two parameters enters into the final analysis.

9.1.1 Sampling Parameters

❶ Sampling conditions

❷ Fixation ① Light microscopy ② Electron microscopy ③ Types of fixative • Formaldehyde • Other

➚

➫ The sampling has to be carried out rapidly to ensure conservation of the nucleic acids. ➫ Everything possible must be done to protect the samples from the action of exogenous RNase and DNase.

➚

➚

➫ Preservation of nucleic acids ➫ Preservation of morphology

➚

➚

➫ An indispensable step ➫ See Chapter 2. ➫ Standard ➫ Often necessary to test the preservation of the nucleic acids by hybridization using a poly (T) probe ➫ Problems: the diffusion of amplified products and the preservation morphology ➫ A compromise between cellular preservation and the diffusion of amplified products

➙ ∼

④ Duration • +

➘

➚

• ++

➙

➙

237

Controls and Problems • +++

❸ Freezing ① Light microscopy • Without fixation

• After fixation ② Electron microscopy ❹ Embedding ① Light microscopy • Paraffin embedding ② Electron microscopy • Before embedding • Without embedding • After embedding • Semithin sections ❺ Storage ① Light microscopy • Frozen sample • Frozen sections • Paraffin-embedded samples • Paraffin-embedded sections ② Electron microscopy • Before embedding • Without embedding • After embedding

➙

➘

➫ Provided that more drastic pretreatments are carried out ➫ Reduction in the diffusion of amplified products

➚➚

➚

➫ Better accessibility of the nucleic acids ➫ If the morphological preservation is poor, a risk of the diffusion of amplified products

➚

➘

➫ Seldom used method

➙

➫ Standard

➙ ∼ ∼ ➙

➫ ➫ ➫ ➫

Standard, for thick sections See Chapter 8. See Chapter 8. For the preembedding method

➙ ➙

➘ ➘

➙

➘

➘

➘

➫ Not recommended; storage of blocks preferable

➙ ∼ ∼

➘

➫ Impossible ➫ See Chapter 8. ➫ See Chapter 8.

➫ −80°C, or liquid nitrogen ➫ Necessary to store the slides in anhydrous conditions ➫ Virtually unlimited conservation period

9.1.2 Pretreatments Parameters

❶ Permeabilization ① Light microscopy • Frozen-tissue sections • Paraffin-embedded sections ② Electron microscopy • Before embedding • Without embedding • After embedding 238

➫ Facilitates the penetration of the reagents and the accessibility of the target nucleic acids ➘

➚

➫ Unnecessary

➘

➘

➫ Not indispensable, but sometimes useful

➙ ∼ ∼

➘

➫ Necessary, with thick sections ➫ See Chapter 8. ➫ See Chapter 8.

9.1 ❷ Deproteinization ① Light microscopy • Frozen-tissue sections • Paraffin-embedded sections ② Electron microscopy • Before embedding • Without embedding • After embedding ❸ Acetylation ❹ Storage ① Light microscopy • Frozen-tissue sections • Paraffin-embedded sections ② Electron microscopy • Before embedding • Without embedding • After embedding

➚

➚

➚ ➙ ∼ ∼ ➘

➘

➙

∼

➙

∼

∼ ∼ ∼

∼

Signal/Background Ratio

➫ Must be light to limit the risk of the diffusion of amplified products due to the destruction of cell structures ➫ Necessary; needs to be sufficient, without, however, affecting morphological preservation ➫ ➫ ➫ ➫ ➫

Necessary, with thick sections See Chapter 8. See Chapter 8. Often unnecessary Blockage of NH2 functions

➫ Although difficult if pretreatments have been carried out, possible in anhydrous conditions ➫ Very difficult if pretreatments have been carried out; preferable to store blocks ➫ Impossible with this step ➫ See Chapter 8. ➫ See Chapter 8.

9.1.3 Reverse Transcription Parameters

❶ Primer ① Type • Specific oligonucleotide • Random oligonucleotides

➙

➙

➫ Standard

➚

➚

• Poly (T)

➚

➙

➫ A risk of nonspecific hybridization of the primers on the large number of fragments of retrotranscribed cDNA ➫ The total cDNA retrotranscribed more accessible

② Concentration • +

➘

➘

• +++ ➙ ③ Hybridization temperature • >42°C ➘ • ≤42°C ➙ • <42°C ➚

➚ ➘ ➙ ➚

➫ A minimal concentration of primers required to avoid the phenomenon of exhaustion ➫ The possibility of nonspecific hybridization ➫ Determination of the primer sequence so that its hybridization temperature is close to the optimal temperature for the activity of the transcriptase (between 40 and 42°C, depending on the brand) 239

Controls and Problems ❷ Enzyme ① Type ② Concentration • + • +++ ❸ Temperature ❹ Duration • <60 min • 60 min • >60 min

➫ See Chapter 4. ➘

➘

➫ The risk of a reduction in the quantity of retrotranscribed cDNA due to exhaustion

➙

➚

➘ ➙ ➚

➘ ➙ ➚

➫ Threshold effect ➫ Standard ➫ Sometimes necessary in electron microscopy (with thick sections)

➙

➙

➫ Standard

➘

➘

➙ ➙

➚ ➙

➫ A reduction in the efficiency of the amplification ➫ The possibility of nonspecific hybridization ➫ Necessary that the two primers hybridize at temperatures that are very close together

➫ Between 40 and 42°C, depending on the brand

9.1.4 PCR Parameters

❶ Primers ① Type • Specific oligonucleotides ② Concentration • + • +++ ③ Temperature ❷ Enzyme ① Type ② Concentration • +

➘

➘

• +++

➙

➚

❸ dNTP ① Concentration • + • +++ ② Type • Direct PCR

➘ ➙ ➙ ➚

➙ ➚ ➙ ➚

➚ ➙

➚ ➙

➘

➙

➚ ➘

➚ ➘

Conjugated dNTP Nonconjugated dNTP • Indirect PCR Nonconjugated dNTP ❹ MgCl2 concentration • Excess • Insufficiency 240

➫ See Section 5.3.3. ➫ A reduction in the efficiency of the amplification ➫ The possibility of nonspecific DNA polymerase activity ➫ Depending on the manufacturer ➫ Standard: 200 µ M ➫ A possible reduction in amplification ➫ An increase in nonspecific reactions ➫ Standard ➫ Necessary to use the recommended respective proportions of conjugated and native dNTP ➫ Indispensable ➫ Indispensable ➫ ➫ ➫ ➫

Indispensable Must be optimized A reduction in the fidelity of the enzyme A reduction in the reaction yield

9.1 ❺ Temperature of the cycles ① Denaturation • <94°C • 94°C • >94°C ② Hybridization (T°H) • T°H ③ Extension • <72°C • 72°C • >72°C ❻ Number of cycles • <20 • 20 • >20 ❼ Final extension • <5 min • 5 min • >5 min

Signal/Background Ratio

➫ Necessary that the thermocycler be capable of producing rapid temperature changes in the sections 0

➘

➫ If the denaturation is partial, neither a PCR nor a RT-PCR can take place ➫ Standard ➫ Sometimes necessary (with thick sections) ➫ See Section 5.5.3. ➫ Threshold effect ➫ Standard ➫ Sometimes necessary to improve the specificity

➙ ➚

➙ ➙

➘ ➙ ➘

➚ ➙ ➘

➘ ➙ ➚

➘ ➙ ➚

➫ A reduction in the amplification reaction ➫ Standard ➫ Sometimes necessary

➘ ➙ ➚

➘ ➙ ➚

➫ Threshold effect ➫ Standard ➫ Sometimes necessary

➘ ➙ ➚

➘ ➙ ➚

➫ Not very effective ➫ Standard ➫ Often necessary ➫ Only in the case of indirect in situ PCR/ RT-PCR

9.1.5 Hybridization Parameters

❶ Probe ① Type • PCR product • Oligonucleotides

② Label • Radioactive • Antigenic ③ Concentration • + • ++ • +++

➙ ➚

➙ ➙

➫ Standard ➫ Chosen from the sequence of the amplified fragment according to the criteria set out in Chapter 6; the two probes must not interhybridize

➚

➚

➘

➘

➫ Background often a problem with radioactive hybridization ➫ A threshold effect in the detection process

➘ ➙ ➙

➘ ➙ ➚

➫ No saturation of targets ➫ Saturation of targets ➫ Increased possibility of nonspecific binding

241

Controls and Problems ❷ Hybridization buffer ① Salt concentration • <600 mM ➘ • 600 mM ➙ • >600 mM ➙ ② Dextran sulfate concentration • <10% ➘ • 10% ➙ • >10% ➙ ③ tRNA, DNA concentration • + ➙ • ++ ➙ • +++ ➙ ④ Detergent • + ➘ • ++ ➙ • +++ ➘ ❸ Temperature • Room temperature ➚ • 37°C ➙ • >40°C ➘ ❹ Duration • <3 h ➘ • 3h ➚ • >5 h ➙ or ➚

➘ ➙ ➚

➫ Reduces the stability of the hybrids ➫ Standard ➫ Increases the stability of the hybrids

➘ ➙ ➘

➫ Reduces the concentration of the probes ➫ Standard ➫ Reduces the possibility of nonspecific bonds ➫ Reduces nonspecific bonds

➙ ➙ ➚ ➙ ➙ ➚

➫ ➫ ➫ ➫

➚ ➙ ➘

➫ Possibility of nonspecific hybridizations ➫ Reduction in nonspecific hybridizations ➫ Possibility of denaturation

➘ ➙ ➚

➫ Limits background ➫ Often sufficient ➫ Possibility of improving the signal

Rarely useful Reduces the penetration of the reagents Not indispensable Favors the diffusion of the hybrids

➫ After PCR and/or hybridization

9.1.6 Washing Parameters

❶ NaCl concentration • +++ •+ ❷ Temperature • Room temperature • >Room temperature ❸ Duration

9.1.7 Detection Parameters

242

➙ ➘

➚ ➘

➫ Stability of the nonspecific hybrids ➫ Denaturation of nonspecific hybrids

➚ ➘ ➘

➚ ➘ ➘

➫ Washing nonspecific hybrids ➫ The denaturation of hybrids ➫ If the washing time too long, the hybrids can become unstable

9.2 ❶ Autoradiographic ① Macroautoradiography ② Microautoradiography

➚ ➚

➙ ➚

❷ Immunocytological ① Method • Direct method • Indirect method ② Label • Fluorescent

➙ ➚

➚ ➙

➘

➚

• Enzymatic • Particle

➙ ➙

➙ ➘

9.2

Sensitivity/Specificity

➫ Standard ➫ The possibility of quantitative estimation ➫ The type of developer, its optimal temperature, and the duration of the reaction will determine whether or not the signal stands out clearly from the background ➫ No penetration of the sections by the reagents ➫ Nonspecific adsorption ➫ An increase in sensitivity ➫ Little used; even after amplification, the signal not strong enough to permit direct observation ➫ Standard ➫ Essentially in electron microscopy

SENSITIVITY/SPECIFICITY

The optimization of a PCR/RT-PCR reaction consists of increasing its sensitivity, i.e., its lower threshold of detection, while ensuring its specificity.

9.2.1 Sampling Parameters

Sensitivity 100

Specificity

50 0

❶ Sampling conditions

❷ Fixation ① Light microscopy

➚

➚

➙

➚

➙

➚

② Electron microscopy ③ Type • Formaldehyde • Others

➙ ~

④ Duration • +

➚

➘

➫ The quality of the sampling, i.e., that of the preservation of the nucleic acids, has an influence on the specificity of the reaction, since breaks can cause parasitic amplifications. ➫ Ensures the preservation of nucleic acids, but also makes them much less accessible ➫ The preservation of morphology ➫ Indispensable step ➫ See Chapter 2. ➫ Standard ➫ Necessary that the fixation conditions be known so that the different reactions can be adapted to them ➫ The shorter the fixation process, the more accessible the nucleic acids 243

Controls and Problems • ++

➙

➙

• +++

➙

➚

➚

➘

➙

➚

❸ Freezing ① Light microscopy • Without fixation

• After fixation ② Electron microscopy ❹ Embedding ① Light microscopy • Paraffin embedding ② Electron microscopy • Before embedding • Without embedding • After embedding • Semithin sections ❺ Storage ① Light microscopy • Frozen samples • Frozen sections • Paraffin-embedded samples • Paraffin-embedded sections ② Electron microscopy • Before embedding • Without embedding • After embedding

➫ A compromise between cellular preservation and the diffusion of the amplified products ➫ In this case, more drastic pretreatments required ➫ A reduction in the diffusion of the amplified products ➫ Better accessibility of the nucleic acids ➫ A risk of increasing the diffusion of the amplified products ➫ Limits the diffusion of the amplified products ➫ Little used

➙

➫ Standard

➙ ~ ~ ➙

➫ The standard, for thick sections ➫ See Chapter 8. ➫ See Chapter 8. ➫ For the preembedding method ➫ If the storage conditions are appropriate, neither the sensitivity nor the specificity of the reaction will be adversely affected.

➙ ➙

➙ ➙

➫ –80°C, or liquid nitrogen ➫ Necessary to store the slides in anhydrous conditions ➫ Virtually no time limit for conservation

➙

➙

➘

➘

➫ Not recommended; storage of blocks preferable

➙ ∼ ∼

➘

➫ Impossible ➫ See Chapter 8. ➫ See Chapter 8.

9.2.2 Pretreatments Parameters

Sensitivity 100

Specificity

50 0

❶ Permeabilization ① Light microscopy • Frozen-tissue sections 244

➫ Facilitates the penetration of the reagents and the accessibility of the target nucleic acids ➙

➘

➫ Unnecessary; and in any case this treatment too aggressive for frozen tissue sections

9.2

Sensitivity/Specificity

➘

➘

➫ Not indispensable; although it may sometimes be useful

➙ ∼ ∼

➘

➫ Necessary (with thick sections) ➫ See Chapter 8. ➫ See Chapter 8. ➫ Facilitates the accessibility of the nucleic acids enclosed in a network of proteins

① Light microscopy • Frozen-tissue sections ➚

➙

➙

➚

➫ Necessary to be only slight if tissue destruction, which in the long run affects specificity, is to be limited ➫ Necessary; must be sufficient to ensure the unmasking of the nucleic acids without, however, affecting the preservation of the morphology

• Paraffin-embedded sections ② Electron microscopy • Before embedding • Without embedding • After embedding ❷ Deproteinization

• Paraffin-embedded sections ② Electron microscopy • Before embedding • Without embedding • After embedding ❸ Acetylation

➙ ∼ ∼ ➘

➘

① Light microscopy • Frozen-tissue sections ➘

➙

➙

➘

❹ Storage

• Paraffin-embedded sections ② Electron microscopy • Before embedding • Without embedding • After embedding

➫ Necessary (with thick sections) ➫ See Chapter 8. ➫ See Chapter 8. ➫ Often unnecessary ➫ Blockage of NH2 functions ➫ The storage of pretreated slides can only reduce the sensitivity and specificity of the reaction ➫ Even in anhydrous conditions, storage after pretreatment a delicate matter ➫ Storage of blocks preferable ➫ Impossible at this stage ➫ See Chapter 8. ➫ See Chapter 8.

∼ ∼

9.2.3 Reverse Transcription Parameters

Sensitivity 100

Specificity

50 0

❶ Primer ① Type • Specific oligonucleotide

➙

➚

➫ Specificity of the PCR determined by that of the RT

245

Controls and Problems • Random oligonucleotides

• Poly (T) ② Concentration • + • +++ ❷ Enzyme ① Type

➚

➘

➚

➘

➘

➙

➚

➘

➫ Numerous fragments of RNA are transcribed into cDNA, which means that the risk of nonspecific hybridization of the primers is increased. ➫ Can increase sensitivity in some cases ➫ Due to a reduction in the efficiency of the RT ➫ The possibility of nonspecific hybridizations ➫ See Chapter 4. ➫ The quality of the enzyme very important for the two criteria in question

② Concentration • +

➘

➚

➙

➘

➘

➘

• +++

➙

➘

❸ Temperature • +

➘

➘

➙

➙

➘ ➙ ➙

➙ ➙ ➙

• +++ ③ Cofactor MgCl2 • +

• +++ ❹ Duration • <60 min • 60 min • >60 min

➫ Due to a reduction in the amount of nonspecific cDNA ➫ A possibility of nonspecific reverse transcription ➫ Due to a reduction in the efficiency of the enzyme ➫ A possibility of nonspecific reverse transcription ➫ Differs according to the manufacturer ➫ Due to a reduction in the efficiency of the enzyme ➫ By reducing the fidelity of the enzyme ➫ A reduction in the efficiency of the enzyme ➫ Standard ➫ A longer incubation time has no effect. Sensitivity depends only on the quality of the enzyme.

9.2.4 PCR Parameters

Sensitivity 100

Specificity

50 0

❶ Primers ① Type • Specific oligonucleotides ② Concentration • + • +++ 246

➙

➙

➫ See Section 5.3.2; necessary to respect the criteria used to determine the specific primers

➘ ➙

➘ ➚

➫ A reduction in the efficiency of the PCR ➫ The possibility of nonspecific hybridization

9.2 ❷ Enzyme ① Type ② Concentration • + • +++ ❸ dNTP ① Concentration • + • +++ ② Type • Direct PCR — Conjugated dNTP — Nonconjugated dNTP • Indirect PCR — Nonconjugated dNTP ❹ Hot start ➎ Temperature of the cycles ① Denaturation • <94°C • 94°C • >94°C ② Hybridization (T°H) • T°H ③ Extension • <72°C • 72°C • >72°C ❻ Number of cycles • <20 • 20 • >20 ❼ Final extension • <5 min • 5 min • >5 min

Sensitivity/Specificity

➫ See Section 5.3.3; the quality of the enzyme is crucial for both sensitivity and specificity ➫ Necessary to follow the manufacturer’s recommendations ➫ A possible reduction in amplification ➫ A possibility of nonspecific DNA polymerase activity

➘ ➙

➚ ➘

➘ ➙ ➙ ➚

➙ ➘ ➙ ➘

➚ ➙

➘ ➙

➫ Standard: 200 µ M ➫ A possible reduction in the amplification ➫ An increase in nonspecific reactions ➫ Standard ➫ The relative proportions of the two types of dNTP is crucial ➫ Indispensable ➫ Indispensable

➘ ➙

➚ ➙

➫ Indispensable

➚

➚

➫ See Section 5.5.2. ➫ The reliability of the thermocycler is important. The sample itself must be at the right temperature.

0 ➙ ➚

➘ ➙ ➙

➚ ➙ ➘

➘ ➙ ➚

➫ A risk of false negatives ➫ Standard ➫ Sometimes necessary ➫ See Section 5.5.3. ➫ A threshold effect ➫ Standard ➫ Sometimes necessary to improve the specificity

➘ ➙ ➚

➘ ➙ ➚

➫ A reduction in the amplification reaction ➫ Standard ➫ Sometimes necessary

➘ ➙ ➚

➘ ➙ ➙

➫ A threshold effect ➫ Standard ➫ Sometimes necessary

➘ ➙ ➚

➘ ➙ ➚

➫ Not very effective ➫ Standard ➫ Often necessary

247

Controls and Problems ➫ Only in the case of indirect reactions

9.2.5 Hybridization Parameters

Sensitivity 100

Specificity

50 0

❶ Probe ① Type • PCR product • Single-stranded DNA • Oligonucleotides ② Label • Radioactive

➙ ➘

➚ ➙

➚

➙

➚

➙

• Antigenic

➘

➘

③ Concentration • + • ++ • +++

➘ ➙ ➙

➚ ➙ ➘

➫ No saturation of the targets ➫ Saturation of the targets ➫ An increase in the possibility of nonspecific bonds

➘ ➙ ➙

➫ Reduces the stability of the hybrids ➫ Standard ➫ Increases the stability of the hybrids

➚ ➙ ➘

➫ Reduces the concentration of the probes ➫ Standard ➫ Reduces the possibility of nonspecific bonds ➫ Reduces the number of nonspecific bonds

❷ Hybridization buffer ① Salt concentration • <600 mM ➘ • 600 mM ➙ • >600 mM ➙ ② Dextran sulfate concentration • <10% ➘ • 10% ➙ • >10% ➚ ③ tRNA, DNA concentrations • + ➙ • ++ ➙ • +++ ➙ ④ Detergent • + ➘ • ++ ➙ • +++ ➘ ❸ Temperature • Room temperature ➚

248

➙ ➙ ➚ ➙ ➙ ➘ ➘

➫ Standard ➫ Limitation of the hybrids to one of the strands of the amplified product ➫ The two probes cannot interhybridize ➫ The sensitivity higher than with antigenic labels ➫ A threshold effect with the detection process ➫ A nonspecific signal, possibly due to endogenous enzymatic activities

➫ Rarely useful ➫ Favors the diffusion of the hybrids ➫ The possibility of nonspecific hybridizations

9.2 • 37°C • >40°C ❹ Duration • <3 h • 3h • >5 h

➙

➙

➘

➚

➫ A reduction in the number of nonspecific hybridizations ➫ Less chance of partial hybrids occurring

➚ ➙ ➘

➫ Less background ➫ Often sufficient ➫ Signal better

➘ ➚ ➙ or ➚

➫ After direct PCR or hybridization

9.2.6 Washing Parameters

Sensitivity/Specificity

Sensitivity 100

Specificity

50 0

❶ NaCl concentration • +++ • + ❷ Temperature • Room temperature • >Room temperature ❸ Duration • +++ • +

➙

➘

➘

➚

➚ ➘

➘ ➚

➘

➚

➙

➘

➫ Stabilization of specific and nonspecific hybrids ➫ Denaturation of nonspecific hybrids ➫ Elimination of nonspecific hybrids ➫ Partial denaturation of hybrids ➫ Must be optimized ➫ If the washing time is too long, possibility that the hybrids can become labile ➫ Limited elimination of partial hybrids

9.2.7 Detection Parameters

Sensitivity 100

Specificity

50 0

❶ Autoradiographic ① Macroautoradiography ② Microautoradiography ❷ Immunocytological ① Method • Direct method • Indirect method ② Label • Fluorescent • Enzymatic • Particle

➚ ➙

➙ ➚

➫ Standard 35 ➫ Cellular resolution with S

➚ ➙

➙ ➚

➫ Standard

➘ ➙ ➙

➙ ➙ ➙

➫ Little used ➫ Standard ➫ Essentially in electron microscopy

249

Controls and Problems

9.2.8 Summary Table

Sample Criterion

Signal

❶ Sampling conditions

➚

❷ Fixation

➚

Background ➚

Sensitivity

Specificity

➚

➚

➙

➚

① Type • Paraformaldehyde

➙

➙

• Other

∼

∼

② Duration • +

➘

➚

➚

➘

• ++

➙

➙

➙

➙

• +++

➙

➘

➙

➚

➚➚

➚

➚

➘

➚

➘

➙

➚

❸ Freezing Light microscopy • Without fixation • After fixation ❹ Embedding ① Paraffin

➙

➙

• Before embedding

➙

➙

• Without embedding

∼

∼

• After embedding

∼

∼

• Semithin sections

➙

➙

② Electron microscopy

❺ Storage ① Light microscopy • Frozen samples

➙

➘

➙

➙

• Frozen sections

➙

➘

➙

➙

• Paraffin-embedded samples

➙

➘

➙

➙

• Paraffin sections

➘

➘

➘

➘

• Before embedding

➙

➘

➙

➙

• Without embedding

∼

∼

• After embedding

∼

∼

② Electron microscopy

250

9.2

Sensitivity/Specificity

Pretreatments Criterion

Signal

Background

Sensitivity

Specificity

• Without embedding

➚

➚

➚

➚

• After paraffin embedding

➚

➙

➚

• Before embedding

➙

➙

➙

• Without embedding

∼

∼

• After embedding

∼

∼

❶ Deproteinization ① Light microscopy

② Electron microscopy

❷ Permeabilization ① Light microscopy • Without embedding

➘

➚

➚

➘

• After paraffin embedding

➘

➘

➙

➘

• Before embedding

➙

➘

➚

➘

• Without embedding

∼

∼

• After embedding

∼

∼

② Electron microscopy

❸ Acetylation

➘

➘

➘

➘

• Without embedding

➙

➘

➙

➚

• After paraffin embedding

➙

➘

➙

➚

• Before embedding

➙

➘

➙

➚

• Without embedding

∼

∼

• After embedding

∼

∼

❹ Prehybridization ① Light microscopy

② Electron microscopy

❺ Storage ① Light microscopy • Without embedding

➙

➘

➘

➙

• After paraffin embedding

➙

➘

➙

➘

• Before embedding

➙

➘

• Without embedding

∼

∼

• After embedding

∼

∼

② Electron microscopy

251

Controls and Problems Reverse transcription Criterion

Signal Background Sensitivity Specificity

❶ Primer ① Type • Specific oligonucleotide

➙

➙

➙

➚

• Nonspecific oligonucleotide

➚

➚

➚

➘

• Poly (T)

➚

➙

➚

➘

•+

➘

➘

➘

➙

• +++

➙

➚

➘

•+

➘

➘

➘

➚

• +++

➙

➚

➙

➘

•+

➘

➘

• +++

➙

➘

•+

➘

➘

• +++

➙

➙

• <60 min

➘

➙

• 60 min

➙

➙

• >60 min

➚

➙

② Concentration

❷ Enzyme ① Type ② Concentration

③ Cofactor

❸ Temperature

❹ Duration

PCR Criterion

Signal Background Sensitivity Specificity

❶ Primer ① Type • Specific oligonucleotide

➙

➙

➙

➙

•+

➘

➘

➘

➚

• +++

➙

➙

➘

② Concentration

252

9.2

Sensitivity/Specificity

❷ Enzyme ① Type ② Concentration •+

➘

➘

➘

➚

• +++

➙

➚

➙

➘

•+

➘

➙

➘

➙

• +++

➙

➚

➙

➘

➙

➙

➙

➙

➘

➘

➚

➘

Conjugated dNTP

➚

➚

➚

➘

Nonconjugated dNTP

➙

➙

➙

➙

➘

➚

➙

➙

➚

➚

❸ dNTP ① Concentration

② Type • Direct PCR

• Indirect PCR Nonconjugated dNTP

➙

➙

❹ Hot start ❺ Temperature of the cycles ① Denaturation • <94°C

0

➘

0

➘

• 94°C

➙

➙

➙

➙

• >94°C

➚

➙

➚

➙

•
➘

➚

➚

➘

• T°H

➙

➙

➙

➙

• >T°H

➘

➘

➘

➚

• <72°C

➘

➘

➘

➘

• 72°C

➙

➙

➙

➙

• >72°C

➚

➚

➚

➚

• <20

➘

➘

➘

➘

• 20

➙

➙

➙

➙

• >20

➚

➚

➚

➙

• <5 min

➘

➘

➘

➘

• 5 min

➙

➙

➙

➙

• >5 min

➚

➚

➚

➚

② Hybridization (T°H)

③ Extension

❻ Number of cycles

❼ Final extension

253

Controls and Problems Hybridization Criterion

Signal Background Sensitivity Specificity

❶ Probe ① Type • PCR product

➙

• Single-stranded DNA

➙

➙

➚

➙

➘

➙

• Oligonucleotide

➚

➙

➚

➙

• Radioactive

➚

➚

➚

➙

• Antigenic

➘

➘

➘

➘

•+

➘

➘

➘

➚

• ++

➙

➙

➙

➙

• +++

➙

➚

➙

➘

• <600 mM

➘

➘

➘

➘

• 600 mM

➙

➙

➙

➙

• >600 mM

➙

➚

➙

➙

• <10%

➘

➘

➘

➚

• 10%

➙

➙

➙

➙

• >10%

➙

➘

➚

➘

•+

➙

➙

➙

➙

• ++

➙

➙

➙

➙

• +++

➙

➚

➙

➚

•+

➘

➙

➘

➙

• ++

➙

➙

➙

➙

• +++

➘

➚

➘

➘

• Room temperature

➚

➚

➚

➘

• 37°C

➙

➙

➙

➙

• >40°C

➘

➘

➘

➚

② Label

③ Concentration

❷ Hybridization buffer ① Salt concentration

② Dextran sulfate concentration

③ tRNA, DNA concentration

④ Detergent

❸ Temperature

254

9.2

Sensitivity/Specificity

❹ Duration • <3 h

➘

➘

➘

➚

• 3h

➚

➙

➚

➙

• >5 h

➙ or ➚

➚

➙ or ➚

➘

Washing Criterion

Signal Background Sensitivity Specificity

❶ NaCl concentration • +++

➙

➚

➙

➘

•+

➘

➘

➘

➚

• Room temperature

➚

➚

➚

➘

• >Room temperature

➘

➘

➘

➚

• +++

➘

➘

➘

➚

➚

➙

➘

❷ Temperature

❸ Duration •+

Detection Criterion

Signal Background Sensitivity Specificity

❶ Autoradiographic • Macroautoradiography

➚

➙

➚

➙

• Microautoradiography

➚

➚

➙

➚

• Direct method

➙

➚

➚

➙

• Indirect method

➚

➙

➙

➚

• Fluorescent

➘

➚

➘

➙

• Enzymatic

➙

➙

➙

➙

• Particle

➙

➘

➙

➙

❷ Immunocytological ① Method

② Label

255

Controls and Problems

9.3

CONTROLS

9.3.1 Tools and Reagents Parameters

Positive

Negative

❶ Primers Search for homologous sequences in a gene bank. ❷ Probes For the hybridization procedure ❸ Specific reagents for amplification Carrying out a liquid-phase PCR for a commonly expressed gene (e.g., actin), using the same reagents ❹ Reverse transcription Carrying out a liquid-phase RT for an RNA expressed in all the types of tissue (e.g., actin), using the same reagents ❺ Immunocytological reaction Using the same reagents to detect a protein that is present in the tissue being studied

256

✔

✔

➫ Significant homologies between sequences can result in the formation of various amplified products, or the amplification of a product that is common to several genes. ➫ Indirect PCR/RT-PCR ➫ The existence of significant homologies between sequences can result in the formation of hybrids that are unwanted but specific to the probes. ➫ The presence of a band on the electrophoresis gel means that all the reagents (i.e., the enzyme, buffer, MgCl2, and nucleotides) are usable for the in situ reaction.

✔

➫ The presence of a band on the electrophoresis gel means that all the reagents (i.e., the enzyme, buffer, cofactor, and nucleotides) are usable for the in situ reaction.

✔

➫ A positive reaction means that all the reagents (i.e., the secondary antibody and the chromogen) are usable for the in situ reaction. ➫ All the reagents (i.e., the primary and/or secondary antibodies, and the chromogen) can be tested to detect a standard in situ hybridization. ➫ If necessary, check that there is no endogenous enzymatic activity.

9.3

Controls

9.3.2 Tissue Parameters

Positive

❶ Positive tissue

Negative

✔

❷ Negative tissue

✔

❸ Internal control

• • • •

Heterogenous culture Coculture Heterogeneous tissue Sections of two different types of tissue ❹ Destruction of the target by treatment • DNase

✔ ✔ ✔ ✔

✔ ✔ ✔ ✔

an enzymatic ✔ ✔

• RNase

➎ Destruction of nontarget nucleic acids by enzymatic treatment • DNase ✔

• RNAse

➫ Indispensable ➫ Tissue or cells known to express the target nucleic acid ➫ Indispensable ➫ Tissue or cells known not to express the target nucleic acid ➫ One of the best controls ➫ Positive and negative cells in the same tissue or culture ➫ Several cell types (e.g., a primary culture) ➫ For example, two cell lines ➫ The most common case ➫ For the same reaction

✔

➫ The breakdown of DNA before the in situ PCR ➫ Necessary to follow the breakdown of RNA by abundant washes before the reverse transcription step ➫ The breakdown of DNA before the in situ RT-PCR, to make sure that there is no amplification of the genomic DNA ➫ The breakdown of RNA before the in situ PCR

9.3.3 Pretreatments Parameters

❶ Fixation

Positive

✔

Negative

➫ An in situ hybridization reaction using poly (T) probes shows that mRNA remains in situ.

257

Controls and Problems ❷ Deproteinization

✔

❸ Permeabilization

✔

❹ The destruction of target nucleic acids. RNAse or DNAse pretreatment

✔

➫ The compatibility of this deproteinization step with the preservation of morphology and of the nucleic acids can be checked by a classical in situ hybridization using a poly (T) probe or a probe corresponding to highly expressed RNA or DNA. ➫ The compatibility of this permeabilization step with the preservation of morphology and that of the nucleic acids can be checked by classical in situ hybridization. ➫ The destruction of these nucleic acids (RNA or DNA) can be checked by classical in situ hybridization.

9.3.4 Reverse Transcription Parameters

Positive

Negative

❶ Omission of the enzyme

✔

❷ Omission of the primer

✔

❸ Omission of the dNTP ❹ Pretreatment with RNase ❺ Pretreatment with DNase

258

✔ ✔

➫ This should abolish the signal. A slightly positive reaction may, however, be observed, corresponding to the signal that would be obtained with hybridization alone. ➫ A simple negative control that gives the same result as before. ➫ This is an unsatisfactory control. The dNTP in the cells is sometimes sufficient to give a positive reaction. ➫ A positive result with the in situ RT-PCR indicates a nonspecific reaction. ➫ No modification of the reaction should be observed if the DNase is RNase-free. A positive result can be seen as the amplification of genomic DNA. ➫ The washes following the pretreatment must be carried out with great care.

9.3

Controls

9.3.5 PCR Parameters

Positive

Negative

❶ Omission of the enzyme

✔

❷ Omission of the primers

✔

❸ Omission of the dNTP

✔

❹ Omission of the labeled dATP ➎ Pretreatment with RNAse • In situ PCR reaction

✔ ✔

• In situ RT-PCR reaction

✔

➏ Pretreatment with DNAse • In situ PCR reaction

✔

• In situ RT-PCR reaction

✔

9.3.6 Hybridization/Washing Parameters

❶ Labeling the probe • Radioactive • Antigenic

Positive

➫ This should abolish the signal, although a slightly positive reaction may be observed, corresponding to the only hybridization of the retrotranscribed RNA and/or the preexisting DNA. ➫ A positive reaction with the in situ PCR/ RT-PCR corresponds, in this case, to the DNA polymerase activity of the enzyme, obtained from breaks in the DNA. In this case the fault may lie with the fixation step. ➫ A positive reaction does not invalidate the in situ PCR/RT-PCR amplification. The dNTP in the tissue may be sufficient to produce amplification. ➫ This is done only in the case of direct in situ PCR/RT-PCR. ➫ No modification of an in situ PCR should be detectable if the RNAse is DNAse-free. ➫ This should abolish the signal. A positive in situ RT-PCR indicates a nonspecific reaction. ➫ This should abolish the signal. A positive in situ PCR indicates a nonspecific reaction. ➫ No modification of the RT-PCR should be observed if the DNAse is RNAse-free. ➫ The washes following the pretreatment must be carried out with great care. ➫ Only with indirect in situ PCR/RT-PCR

Negative

➫ Determination of the specific activity ➫ Dot on membrane

259

Controls and Problems ❷ Omission of the probe

✔

❸ Hybridization with a heterologous probe of the same type ❹ Action on the efficiency of the hybridization reaction ➎ Action on the stringency of the washes

✔

➫ If the reaction is positive, the presence of an endogenous label (e.g., biotin, or an enzyme) must be suspected. ➫ The specificity of the detection of the amplified product ➫ A variation in the hybridization temperature induces a modification of the signal. ➫ A variation in the salt concentration or the temperature of the washes induces a modification of the signal.

9.3.7 Detection Parameters

Positive

❶ Autoradiographic ❷ Immunohistological • Omission of the primary antibody • Omission of the conjugated antibody

❸ Omission of the fluorescent label or chromogen

Negative

✔

➫ Determination of the background

✔

➫ A negative reaction

✔

➫ A search for endogenous activity: • Endogenous biotin • Endogenous peroxidase • Endogenous alkaline phosphatase All positive reactions can be inhibited by specific chemical compounds (e.g., hydrogen peroxide or levamisol). ➫ A positive reaction reveals the presence of fluorescence or an induced stain.

✔

9.3.8 Results Parameters

❶ Signal/background ratio

260

Positive

✔

Negative

➫ Indispensable (see Section 9.3.2)

9.3 ❷ Reproducibility

✔

❸ Extraction of the amplified product from a section

✔

❹ Reaction medium

✔

Controls

➫ A similar location of the signal in two adjacent sections, or a section from another sample processed in the same conditions. ➫ With migration on a gel, a band should appear at a predetermined position on the column. ➫ Sample the reaction medium for the amplification step, and if, after electrophoresis, a band corresponding to the size of the amplified product appears, this means that a large amount of diffusion has taken place, in which case the possibility of a false positive must be considered, and the experimental conditions revised (see Section 9.4).

9.3.9 Validation by Other Techniques Parameters

❶ Liquid-phase PCR ❷ Liquid-phase RT-PCR ❸ Immunocytology ❹ Transgenic animals ➎ “Knockout” animals

Positive

Negative

➫ Demonstration of the presence or absence of the target DNA in the tissue under consideration ➫ Demonstration of the presence or absence of the target RNA in the tissue under consideration ➫ Detection of the protein transcribed from the target nucleic acid ➫ Overexpression of the gene being sought ➫ Suppression of the gene being sought

261

Controls and Problems

9.3.10

Summary Table Methods

Tissue

Controls

PCR

RT-PCR

Direct

Indirect

Direct

Indirect

Positive

+

+

+

+

Negative

0

0

0

0

Internal control

+

+

+

+

Adjacent sections

+

+

+

+

Destruction of the targets

0

0

0

0

Fixation

+/−

+/−

+/−

+/−

Deproteinization

+/−

+/−

+/−

+/−

Permeabilization

+/−

+/−

+/−

+/−

+

+

Enzyme

0

0

Primer

0

0

dNTP

+/−

+/−

Modification

Pretreatments

Reverse transcription

Omission

Liquid-phase reaction

Amplification +

+

+

+

Taq DNA polymerase

0

0

0

0

Primers

0

0

0

0

Nonconjugated dNTP

0

0

0

0

Conjugated dNTP

0

Omission

Liquid-phase reaction

0

+/−

+/−

+/−

+/−

Positive tissue

+

+

+

+

Omission of probes

0

0

0

0

Heterologous probe

0

0

0

0

Stringency of washes

+/−

+/−

+/−

+/−

+

+

+

+

Stringency of washes Hybridization

Detection Positive control

262

Omission

9.4

False Positives

Primary antibody

0

0

0

0

Secondary antibody

0

0

0

0

Chromogen

0

0

0

0

+

+

+

+

0

0

0

0

Results Reproducibility Signal/background ratio Reaction medium Other techniques +

9.4

Positive result

+/−

Variable result

0

Negative result

FALSE POSITIVES

False positives are due to suspect controls, which do not allow the signal to be identified as specific to the amplification of the sequence of interest. The causes are relatively few in number:

• The nonspecific synthesis of the sequence of interest can result from: • The nonspecific incorporation of the label • The repair of cellular DNA • Nonspecific amplification • Nonspecific hybridization of the primers • Amplification of genomic DNA • Diffusion of the amplified products • External contamination

➫ This is a significant risk in in situ PCR/ RT-PCR. ➫ See Section 9.3.9. ➫ It is not always a problem of specificity strictly speaking, but of a parallel reaction brought about in the course of the in situ amplification. ➫ This is possible when all the different types of tissue are positive, including the tissue used as a negative control. ➫ This is possible only with direct reactions. ➫ From fixation to pretreatments, there are numerous possible causes for breaks in DNA. ➫ Taq DNA polymerase becomes active at ≥40°C, attaching to a 3′ hydoxylated end. ➫ This mostly concerns the primers that define the PCR. ➫ This risk should not be overlooked (see Chapter 5). ➫ Among the different causes of false positive, this is the most difficult to detect. ➫ This is normally a low risk.

It is also necessary to check for false positives that might result from less fundamental causes: • Problems due to the specificity of the reagents • Nonspecific detections

• Handling errors • Artifacts, etc.

➫ These can be due to endogenous enzymatic activity and to nonspecific tools (antibodies, chromogens). ➫ Recommence the reaction. ➫ Recommence the reaction. 263

Controls and Problems

9.4.1 Nonspecific Incorporation of Labels The labels are carried either by: • Labeled dNTP, or • Labeled primers, used in direct in situ PCR or RT-PCR 9.4.1.1

➫ Essentially dUTP or dATP (see Section 5.3.1) ➫ See Section 5.3.2.7.

Definition of the problem

This may be the problem when a signal: • Appears in tissue defined as negative • Is found in all the cells in the section being studied • Is not inhibited by a control carried out in the absence of primers 9.4.1.2

➫ See Section 9.3. ➫ An absence of negative cells ➫ See Section 9.3.

Causes

There are two main causes: • Nonspecific synthesis • The presence of residual dNTP or labeled primers 9.4.1.3

➫ A consequence of the nonspecific hybridization of primers ➫ A consequence of insufficient washing

Solutions

There are several possible solutions, depending on the strength of the signal. • Use the indirect method. • Increase the stringency and duration of the washes. • Increase the hybridization temperature of the primers.

➫ The disappearance of the labeled primers or dNTP eliminates this problem. ➫ This eliminates the labeled primers or dNTP not incorporated into the amplified products. ➫ This potentially reduces nonspecific amplifications.

9.4.2 Repair of Cellular DNA Nucleic acids undergo modifications, destruction, and repair. And, in fact, breaks are sometimes used as markers (e.g., apoptosis). The repair of breaks in the presence of dNTP and an enzyme is thus a phenomenon that must not be overlooked.

264

➫ Breaks can be revealed by specific labeling (i.e., in situ 3′ extension). ➫ The risk concerns only direct PCR/ RT-PCR reactions. ➫ The label must be carried by dNTP. The problem does not exist if the label is carried by the primer.

9.4 9.4.2.1

Definition of the problem

• There is an amplified signal, but also nonspecific labeling. • In the absence of an enzyme, these two signals disappear. • In the absence of primers, on the other hand, this nonspecific labeling persists, whereas the amplified signal disappears. 9.4.2.2

➫ DNA polymerase acts both in the amplification reaction and in the repair of breaks. ➫ The signals do not appear in the absence of enzyme. ➫ No amplification takes place in this case, but the repair of breaks remains possible.

Causes

Taq DNA polymerase acts on breaks in DNA. 9.4.2.3

False Positives

➫ This property can be used for in situ labeling.

Solutions

• The destruction of the genomic DNA before amplification • An indirect reaction

➫ For pretreatment with DNase, see Section 3.7.3. ➫ This repair can also take place during an indirect reaction, but is not detected during the hybridization step.

9.4.3 Nonspecific Synthesis Any synthesis not corresponding to the amplified product is considered to be nonspecific.

➫ Detected by suspect controls

9.4.3.1

➫ Note: This signal may be superimposed on the specific signal. ➫ The polymerase activity disappears.

Definition of the problem

• This signal disappears if the enzyme is omitted. • Negative tissue appears positive. • There is no internal control. • The signal is not reproducible on two adjacent sections. 9.4.3.2

➫ This test is the most convincing. ➫ This synthesis can appear in a random way in any of the cells. ➫ This nonspecific signal can be superimposed on a specific signal.

Causes

This type of synthesis can have different causes: • Amplification common to several genes

• Nonspecific hybridization of the primers. • Repair of breaks with: • Incorporation of labeled nucleotides • Hybridization of certain random sequence regions with probes intended for the detection of the amplified products.

➫ They are given in decreasing order of importance. ➫ Verification of the absence of homology between the primer sequences and one or more sequences within the genome ➫ Particularly high risk ➫ The synthesis of random sequences ➫ Labeling during synthesis (direct method) ➫ Minimal risk

265

Controls and Problems 9.4.3.3

Solutions

• An increase in the hybridization temperature of the primers during the PCR cycles • An indirect reaction • An increase in the hybridization temperature of the probes

➫ An increase in the specificity of the PCR step ➫ Only the amplified products detected by the hybridization step ➫ An increase in the specificity of the detection step

9.4.4 Nonspecific Hybridization of the Primers This is a crucial phase in the reverse transcription step and the PCR cycle, as the hybridization of the primers determines the specificity of the reaction. 9.4.4.1

Definition of the problem

• Tissue that is normally negative is in fact positive. • In heterogeneous tissue, all the cells are positive. • Stringent washes abolish this signal. 9.4.4.2

➫ One of the best controls for detecting false positives ➫ In the case of an internal control ➫ One solution to the problem

Causes

• Nonspecific hybridization of the primer specific during the RT step can result in the synthesis of DNA of different sizes. • Nonspecific hybridization of the primers for the PCR step leads to the synthesis of strands of DNA that do not correspond to the nucleic acid sequence of interest. 9.4.4.3

➫ Hybridization errors result in nonspecific syntheses.

➫ The efficiency of the PCR will then be lower, but this lack of specificity cannot, on its own, explain a false positive. ➫ Their nucleotide sequences and Tm may be responsible for this.

Solutions

• Change the primers.

• Increase the hybridization temperature(s) of the PCR cycle. • Increase the stringency of the washes to denature partial hybrids.

➫ Do this where only the amplification is nonspecific. ➫ Check the potential complementarity of their sequences against a gene bank. ➫ Check that the two primers hybridize at very similar temperatures. ➫ See Section 5.5.7.

9.4.5 Amplification of Genomic DNA This is the most common type of false positive. The signal is generally limited to the nuclear compartment, and can be present in any of the cells.

266

➫ All the cells in an organism have the same genome. ➫ This risk is high, and the experimenter must be very careful about the choice of the primers and their position on the gene in relation to the introns.

9.4 9.4.5.1

Definition of the problem

• All the nuclei are positive.

• No reaction-extinction control is conclusive. • The controls for the reverse transcription step remain positive. • The controls for the amplification step are correct. 9.4.5.2

➫ It must also be remembered that the nuclear signal can have different origins (e.g., a nuclear virus, a detection problem, or the diffusion of amplified products into the nuclear compartment). ➫ See Section 9.3. ➫ See Section 9.3. ➫ The reverse transcription can be effective, and can lead to specific amplification. ➫ See Section 9.3. ➫ This type of amplification is specific.

Causes

• There are at least two copies of the gene of interest in the genomic DNA. • The primers are incorrectly positioned on the structure of the gene. 9.4.5.3

False Positives

➫ Some authors claim that amplification can take place with just one copy.

Solutions

• A change of primers, so that this type of genomic amplification becomes impossible • Verification of the new primers • Destruction of the genomic DNA before amplification

➫ See Section 5.3.3.2. ➫ By liquid-phase PCR ➫ Pretreatment with DNase (see Section 3.7.3)

9.4.6 Diffusion of Amplified Products The amplified products are obtained from sequences of nucleic acids in cell structures consolidated by fixation. Given the permeabilization necessary to the penetration of tools and reagents, it is important to check that an amplified product has not diffused, first into adjacent cells, and second into the reaction medium, where it can be eliminated by washing. It is sometimes said that the multiplication of the amplified products could cause them to move away from the original target sequence. 9.4.6.1

➫ In theory, the risk of false positives resulting from the diffusion of an amplified product is considerable, but in practice it seems minor.

➫ This remains hypothetical.

Definition of the problem

• The signal diffuses along a decreasing gradient from the most to the least positive cells. • The amplification controls are positive. • The internal control is negative.

➫ There can be so much diffusion that all the tissue appears positive. ➫ See Section 9.3. ➫ This is the best control for evaluating this particular risk.

267

Controls and Problems 9.4.6.2

Causes

• Partial or total destruction of cell or tissue structures • Faulty fixation 9.4.6.3

Solutions

• An improvement in the fixation conditions • An increase in the stringency of the washes

• A reduction in the pretreatments, and in particular the permeabilization and deproteinization steps.

9.4.7 External Contamination External contamination can produce a positive result in tissue. However, it depends on such an unlikely combination of preconditions that for practical purposes it does not constitute a risk of false positives. 9.4.7.1

➫ Poor fixation, or overaggressive pretreatments ➫ The most common cause

➫ To reinforce the cell and tissue structure ➫ The amplified products that have diffused are seldom if ever attached to cell or tissue structures, and are easy to eliminate. ➫ Making sure that the PCR reaction is not, however, inhibited; a satisfactory compromise may be difficult to find ➫ This is a minor theoretical risk, as the tissue is an entity in itself. ➫ It can occur at any level.

Definition of the problem

• A homogeneous signal is observed over the whole section, or along a gradient which decreases from the periphery inward. • All the controls of specificity are positive, but the negative control is positive. • The internal control allows this risk to be evaluated.

➫ Contamination by the external medium. ➫ See Section 9.3. ➫ The signal is specific. Only internal controls (i.e., negative cells) can provide hard evidence of contamination.

• The result is not reproducible from one slide to another. 9.4.7.2

Causes

The presence of target sequences external to the sample, which contaminate: • Equipment • Sections • Solutions 9.4.7.3

Solutions

• Replacement of all primers, enzymes, reagents, etc. • Replacement of all the minor equipment

268

➫ Sequences deriving from previous reactions ➫ While they are being cut ➫ While they are being prepared ➫ If the cause is not identified, all possible precautions must be taken. ➫ Pipettes, single-use articles, etc.

9.5 ➫ ➫ ➫ ➫

• Replacement of all the preparations • Decontamination of the work surfaces • Decontamination of the thermocycler

False Negatives

Fixatives, and in particular the buffers The laboratory Separation of the working areas With some models, this is easy

Amplification of the genomic DNA

Diffusion of the amplified products

External contamination

+

+

+

+

+

+

+

Internal control

+

+

+

+

+

+

+

Controls

Nonspecific hybridization of the primers

Nonspecific synthesis

Negative

Causes

Nonspecific incorporation of the label

Cellular DNA repair

9.4.8 Summary Table

Tissue

Positive

Adjacent sections Destruction of the targets

+

Pretreatments

+

+

+

+

+

+

+ +

Taq DNA polymerase Omission

+

Reverse transcription

+

+

Primers

+

+

+

+

+

+

+

+ +

Hybridization Detection

Stringency of the washing

+

+

False positive Controls to determine the cause of a false positive.

9.5

FALSE NEGATIVES

These are characterized by the result of a negative in situ PCR/RT-PCR on tissue known to be positive. False negatives are much less frequent than false positives. Among their causes are the following: • The destruction of target sequences • Fixation problems • Digestion problems

➫ All the controls carried out in the liquid phase must be positive. ➫ The aim of this technique is to identify nucleic acid sequences that are very weakly expressed, and can thus very easily be lost during: • The preparation of the sample • The stabilization of the structures • The pretreatments 269

Controls and Problems • Flaws in the reverse transcription process

• Flaws in the amplification process

• Overabundant washes The first option is to increase the number of cycles. One must, however, exclude false negatives caused, for example, by: • The quality of the reagents • Handling errors • Artifacts

➫ These are due to a problem with the hybridization of the primer, or a malfunctioning enzyme. ➫ These are due to a problem with the hybridization of the primer, or a malfunctioning enzyme. • Elimination of the amplified products ➫ With more than 30 cycles, it is improbable that a specific result will be obtained. ➫ See Section 9.3.

➫ Recommence the reaction. ➫ Recommence the reaction.

9.5.1 Destruction of Target Sequences These sequences, of which there are only a few copies in a handful of cells, can be destroyed, in particular, during sampling or pretreatment if certain conditions are not satisfied. 9.5.1.1

Definition of the problem

• The absence of a signal in the positive control tissue. • The amplification reaction carried out on the same sample after the extraction of the nucleic acids is: — Either equally negative, or — Positive

9.5.1.2

• Storage of samples

➫ The sample must be responsible. ➫ The in situ manipulation must be responsible.

➫ Intracellular autolysis ➫ Contamination by RNase or DNase from the equipment or the operator ➫ A problem, in particular, with frozen samples

Solutions

• The sampling conditions and protocol will need to be reviewed. • Check for the presence of RNA before carrying out an in situ RT-PCR reaction by in situ hybridization with a poly (T) probe.

270

➫ The only evidence of false negatives in situ

Causes

• Overlengthy sampling times • Nonsterile sampling conditions

9.5.1.3

➫ This is a major theoretical risk, in particular for RNA sequences (i.e., for RT-PCR methods). ➫ If the sampling is not rapid enough, intracellular autolysis may occur.

➫ If the sample comes from a retrospective series, look at the way the sample was processed. ➫ It is possible that the sample was not processed in conditions favorable to the conservation of nucleic acids.

9.5

False Negatives

9.5.2 Problems Related to the Fixation Process This step is indispensable to the morphological preservation and stabilization of structures, but it often results in the partial destruction of the target sequences.

9.5.2.1

Definition of the problem

• Signal is absent in the positive control tissue. • A positive amplification reaction is obtained on the same sample after the extraction of the nucleic acid. • The morphology of the tissue is excellent.

• The fixation conditions and/or the type of fixative are not known. • Carry out a hybridization with a Poly (T) probe.

9.5.2.2

Causes

• Target sequences are lost. • The confinement of target sequences in a molecular framework is more or less marked according to the duration of the fixation process. • The target sequences are modified by the fixative.

9.5.2.3

➫ Prolonged fixation can destroy or mask target sequences, while, if it is reduced to the minimum, it results in false positives (see Section 9.4). A compromise must be found for each type of tissue studied, on a case-by-case basis.

Solutions

• Check the fixation conditions. • Change the fixation conditions. • Change the pretreatments.

➫ The only evidence of false negatives in situ ➫ Positive PCR/RT-PCR in liquid phase ➫ Necessary to consider the trade-off between morphological preservation and the preservation of target nucleic acids (see Chapters 2 and 3). ➫ See Chapter 2; some fixatives incompatible ➫ If this is negative, there is no more mRNA in the tissue, and it is likely that the sequence of interest has also disappeared (see Section 2.1.3.3). ➫ These are often simple, and result from a lack of information about how the sample was prepared. ➫ This is by diffusion, if the fixation is insufficient. ➫ The target sequences are masked, and are difficult to access. ➫ Some fixatives cause breaks in the target sequences, others modifications of the bases (see Chapter 2). ➫ These are generally very simple. ➫ Indispensable ➫ If possible, apply the standard conditions (see Chapter 2). ➫ This reduces the cross-linkages created by the fixative between the nucleic acids and the proteins (see Chapter 3).

271

Controls and Problems

9.5.3 Problems Related to Proteasic Digestion This step is indispensable to making the nucleic acid accessible and facilitating the penetration of the tools and reagents. 9.5.3.1

Definition of the problem

• An absence of signal in the positive control tissue • A positive result for an amplification reaction carried out on a sample after the extraction of the nucleic acid • Poor preservation of tissue morphology • The reaction mixture analyzed after in situ amplification: — A band of the expected size appears on the electrophoresis gel — A band of the expected size appears after a PCR or a “nested” PCR 9.5.3.2

➫ The only evidence of false negatives in situ ➫ Positive PCR/RT-PCR in liquid phase ➫ Often results in poor preservation of the target nucleic acid ➫ This test demonstrates the diffusion of the amplified product. ➫ The diffusion is minimal, and does not explain the false negative.

Causes

• The loss of target sequences

• The loss of cDNA • The loss of amplified products

9.5.3.3

➫ Risks due to pretreatments (see Chapter 3)

➫ By diffusion from tissue made overpermeable by pretreatments (e.g., deproteinization or permeabilization) ➫ By diffusion during washing after reverse transcription ➫ By diffusion during washing after amplification

Solutions

• Cut down the deproteinization step by reducing: — The proteinase K concentration — The incubation time • Change the fixation conditions. • Reduce, or cut out altogether, the permeabilization step.

➫ Conservation of the target sequences in the cell ➫ May be necessary to change the brand or the batch ➫ Step needs to be optimized (see Section 3.5.2.1) ➫ Conservation of the cell structures ➫ A reduction in the diffusion of the amplified products

9.5.4 Reverse Transcription Problems This is a necessary step that precedes the amplification step. Without it, no amplification of RNA would be possible.

272

➫ Limited to in situ RT-PCR methods ➫ This is the most difficult step to control, and is probably the one that gives rise to the largest number of unexplained negative reactions.

9.5 9.5.4.1

Definition of the problem

The possibility of reverse transcription problems is generally considered only after other potential problems have been ruled out. • An absence of signal in the positive control tissue • A control carried out in the liquid phase after the extraction of the nucleic acid from the same sample: — Positive reaction

— Negative reaction

9.5.4.2

Causes

• The primer • The enzyme

• The cofactor of the enzyme • The reagents • The experimental conditions

9.5.4.3

False Negatives

➫ This is the only evidence of false negatives in situ.

➫ The RT conditions are good (enzyme, concentration); the problem has to do with the in situ adaptation. This is a genuine false negative. ➫ The problem with the RT will be difficult to identify. ➫ There are a number of potential causes. ➫ Essentially a problem of concentration ➫ Essentially a problem of concentration, although its effectiveness, age, and storage conditions should also be checked ➫ Necessary to optimize its concentration ➫ Unlikely to be responsible if the reaction is positive in the liquid phase ➫ That is, the temperature, the duration, and the programming of the thermocycler

Solutions

• Increase the concentrations of the different reagents. • Modify the thermocycler settings.

➫ That is, the enzyme, the primer, and the cofactor ➫ Lower the hybridization temperature, even though this involves the risk of a nonspecific signal being generated.

9.5.5 Amplification Problems

➫ The usual risks, especially for an inexperienced operator, as all the steps must be optimized

There are a number of potential sources of problems.

➫ The equipment, programming, handling, reaction medium, reagents, primers

9.5.5.1

Definition of the problem

• An absence of signal in the positive control tissue • A control carried out on the same sample after the extraction of the nucleic acid: — Positive reaction

➫ This is the only evidence of false negatives in situ. ➫ The in situ adaptation alone must be responsible. This is a genuine false negative. 273

Controls and Problems — Negative reaction

• The reaction remains negative after several assays, whereas other reactions were positive. 9.5.5.2

Causes

• A malfunctioning of the thermocycler

• A handling error • Incompatible hybridization temperatures of the two primers • Insufficient amount of enzyme • The MgCl2 concentration too low • PCR chamber adsorbing the reagents 9.5.5.3

➫ Numerous, and often cumulative ➫ The temperatures of the sections do not correspond to the indicated temperatures. ➫ The programming of the temperatures and durations of the different phases of the cycle is faulty. ➫ There are bubbles in the reaction medium. ➫ The hybridization phase of the cycle does not take place properly. ➫ The enzyme is adsorbed, which means that no amplification occurs. ➫ The cofactor is adsorbed, which means that no amplification occurs. ➫ No amplification can take place.

Solutions

• It is difficult to calibrate the thermocycler. It is, however, possible to test the temperature of the three fundamental stages of the PCR cycle. — Denaturation temperature

— Hybridization temperature

— Extension temperature • Repeat the reaction to eliminate risks resulting from the experimenter. • Check the Tm of the primers.

• Make a range of concentrations of the enzyme and of MgCl2. • Pretreat the components of the reaction chamber.

274

➫ It is not possible to determine the origin of the problem. It will be necessary to carry out a PCR on a strongly expressed target sequence to test the experimental conditions, the reagents, the thermocycler, etc. ➫ The lack of experience of the experimenter can be ruled out.

➫ Test another thermocycler if possible. ➫ Test with a known positive reaction in liquid phase. ➫ Amplification of strongly expressed test DNA. A genuinely negative result may indicate that the denaturation temperature has not been attained. ➫ Hybridization using an oligonucleotide probe with a low Tm; a negative result indicates that the temperature was too high. ➫ Whatever the temperature difference, the amplification reaction is weak, but not negative. ➫ These reactions are very sensitive, and a lack of attention often produces a negative (or false-positive) result. ➫ See Chapter 4. ➫ It is possible to extend the primer with the lowest Tm so as to increase it. ➫ See Chapter 5. ➫ Commercial products are ready to use. If cover slides are used, it is recommended that they be siliconized and sterilized (see Appendix A3.2).

9.5

False Negatives

9.5.6 The Stringency of the Washes

➫ The risk is all the higher, as the amplification is weak.

The stringency of the washes must be adapted to the experiments on a case-by-case basis. It should be increased only to reduce nonspecific signals.

➫ To adjust the stringency, it is preferable to choose conditions that give a signal, even if there is a significant amount of background, which can be eliminated in a number of ways. To start out from a negative result is always more difficult.

9.5.6.1

Definition of the Problem

• An absence of signal in the positive-control tissue. • A positive amplification reaction carried out on the same sample after the extraction of the nucleic acid. • The morphological preservation is not satisfactory. • A nested PCR carried out on an aliquot of the reaction mixture sampled after the amplification should be positive. 9.5.6.2

Causes

• Elimination of the amplified products. 9.5.6.3

➫ The only evidence of false negatives in situ. ➫ Positive PCR/RT-PCR in liquid phase. ➫ Poor preservation of the structures favors the elimination of the amplified products. ➫ The amplification has in fact taken place.

➫ Numerous, and often cumulative. ➫ Especially if the amplification is weak.

Solution

• Reduce the stringency of the washes.

➫ Limit the washes to one or two baths of 2X SSC.

9.5.7 Detection Problems

➫ Rare.

Detection is now a well-worked-out step, both in immunocytology and autoradiography.

➫ It must be checked on a positive model.

9.5.7.1

Definition of the problem

• An absence of signal in the positive-control tissue. • A positive amplification reaction carried out on a sample after the extraction of the nucleic acids. • There is no background. 9.5.7.2

Causes

• Handling errors. • Antibodies, chromogens, or nuclear emulsions beyond their expiration date.

➫ The only evidence of false negatives in situ. ➫ PCR/RT-PCR in positive liquid phase. ➫ There may be exceptions. ➫ Occasional. ➫ The most common. ➫ The detection step was not carried out.

275

Controls and Problems 9.5.7.3

Solutions

• Review the protocol. • Check the reagents. • Recommence the detection process with another protocol.

➫ See Chapter 7. ➫ Using another technique (e.g., immunocytology) ➫ See Chapter 7.

Pretreatments

Reversetranscription problems

Amplification problems

Stringency of the washes

Tissue

Controls

Fixation

Causes

Destruction of the target sequences

9.5.8 Summary Table

−

−

−

−

−

−

−

−

−

−

−

−

−

−

−

−

−

−

Positive Negative Internal control Adjacent sections Destruction of the targets Pretreatments

Omission

Taq DNA polymerase Reverse transcription Primers Hybridization Detection Stringency of the washes −

Negative control

Controls to determine the cause of a false positive.

276

Chapter 10 Typical Protocols

Contents

CONTENTS 10.1 Direct in Situ PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2 Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.2 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . 10.1.2.2.1 Cell Cultures . . . . . . . . . . . . . . . . . . . . 10.1.2.2.2 Frozen Fixed Tissue . . . . . . . . . . . . . . . 10.1.2.2.3 Fixed Paraffin-Embedded Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.3 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.3.1 Cell Cultures and Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.3.2 Paraffin-Embedded Fixed Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.4 In Situ Amplification . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.5 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2.6 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Indirect in Situ PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2 Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.2 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . 10.2.2.3 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.4 In Situ Amplification . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.5 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.5.1 Labeling Probes by Extension of the 3′ End . . . . . . . . . . . . . . . . . . . . 10.2.2.5.2 Hybridization . . . . . . . . . . . . . . . . . . . . 10.2.2.6 Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2.7 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Indirect in Situ RT-PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2 Typical Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.2 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . 10.3.2.3 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.4 DNase Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.5 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.6 In Situ Amplification . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.7 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.8 Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2.9 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Direct in Situ RT-PCR—Special Case: Cell Suspension. . . . . . . . . . . . 10.4.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.4.2 Typical Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

281 281 282 282 282 282 283 283 284 284 285 285 287 288 289 289 290 290 290 290 291 291 291 292 293 294 294 295 295 295 296 296 296 297 297 298 299 300 300 301 302 302 279

Typical Protocols 10.4.2.2 10.4.2.3 10.4.2.4 10.4.2.5

Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . Amplification with Labeled Primers . . . . . . . . . . . . 10.4.2.5.1 Fluorescent Primers . . . . . . . . . . . . . . . 10.4.2.5.2 Biotinylated Primers . . . . . . . . . . . . . . 10.4.2.6 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 Indirect in Situ RT-PCR on Vegetable Tissue Using Floating Vibratome Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2 Typical Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.3 Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.4 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.5 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.6 Amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.7 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.8 Immunocytological Detection . . . . . . . . . . . . . . . . . 10.5.2.9 Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.2.10 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 Indirect in Situ RT-PCR Using Electron Microscopy . . . . . . . . . . . . . . 10.6.1 Diagram of the Different Steps . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2 Typical Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.3 Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.4 Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.5 Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.6 Amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.7 Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.8 Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.9 Ultramicrotomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.10 Immunocytological Detection . . . . . . . . . . . . . . . . . 10.6.2.11 Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6.2.12 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

280

303 304 304 305 305 306 308 308 309 310 310 310 311 311 311 312 313 314 314 315 315 316 316 316 317 317 318 318 318 319 320 321 321 321 321

10.1 When using in situ PCR and RT-PCR methods, the idea of “typical protocols” represents something of a challenge. The protocols given in this chapter should help beginners save time and avoid the main pitfalls. These “recipes” do, however, need to be adapted and adjusted to the researcher’s personal objectives, and in the end it is only through a process of trial and error that satisfactory results will be obtained. A positive result cannot be taken for granted unless controls are carried out.

Direct in Situ PCR

➫ See Chapter 9.

10.1 DIRECT IN SITU PCR

➫ See Chapter 11, Figures 11.1 and 11.2.

Direct in situ PCR is essentially used to detect viral DNA or identify particular genes in cultured cells, frozen fixed tissue, or paraffin-embedded tissue sections.

➫ Gloves must be worn. All the products must be RNase-free, the solutions must be prepared in DEPC water (see Appendix B1.2), and all equipment must be sterilized (see Appendix A2).

10.1.1 Diagram of the Different Steps

281

Typical Protocols

10.1.2 Typical Protocols 10.1.2.1

Solutions

BCIP 0.4 mM biotin-14-dATP Blocking agents 10 mM dTTP, dCTP, dGTP, dATP Ethanol 100° (−20°C) Ethanol 70°, 95°, 100° Isopentane Liquid nitrogen 50 mM MgCl2 9‰ NaCl NBT Paraffin 4% paraformaldehyde 10X PCR buffer 0.1 M phosphate buffer 10 µM anti-sense primer 10 µM sense primer Proteinase K Streptavidin conjuguated to alkaline phosphatase 5 U/µl Taq DNA polymerase 0.1 M Tris–HCl buffer Tris–HCl/CaCl2 buffer Tris–HCl/NaCl buffer Tris–HCl/NaCl/MgCl2 buffer DEPC water Sterile water Xylene or methyl cyclohexane 10.1.2.2

Preparation of samples

10.1.2.2.1 CELL CULTURES ❶ Preparation of cells 6 10 cells/30 ml of medium, supplemented with 5 to 10% calf fetal serum and antibiotics, are cultivated on pretreated slides in culture boxes. The cells are confluent for: 2 days ❷ Washing 0.1 M phosphate buffer 2 × 5 min ❸ Fixation a. Incubate the sections: • 4% paraformaldehyde 15 min in phosphate buffer

282

➫ See Appendix B6.2.2.1. ➫ See Appendix B6.2.1. ➫ dNTP

➫ See Appendix B2.12. ➫ See Appendix B2.19. ➫ See Appendix B6.2.2.1. ➫ ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B4.3.2. Use the one that is supplied with the enzyme. See Appendix B3.4.1. See Section 5.3.2. See Section 5.3.2. See Appendix B2.14.1. Or conjugated anti-biotin IgG

➫ ➫ ➫ ➫ ➫ ➫

See Appendix B2.21. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.6. See Appendix B1.2. See Appendix B1.1.

➫ See Chapter 2.

➫ 100 µg/ml penicillin, 100 U/ml streptomycin ➫ See Appendix A3 or on Perkin-Elmer slides. ➫ Time variable according to the type of cell. ➫ This process requires great care, as the cells are delicate and risk becoming unstuck. ➫ It is important to pay attention to the fixation time: insufficient fixation leads to the diffusion of products, whereas excessively long fixation hinders the penetration of the reagents.

10.1 b. Rinse: • 0.1 M phosphate buffer ❹ Dehydration • Ethanol 70°, 95°, 100° ❺ Drying • Under a hood 10.1.2.2.2 FROZEN FIXED TISSUE ❶ Tissue preparation a. Fix: • 4% paraformaldehyde b. Rinse: • 0.1 M phosphate buffer • 30% sucrose in 0.1 M phosphate buffer

c. Freeze: • In liquid nitrogen vapor, or • In isopentane cooled in liquid nitrogen. d. Store.

Direct in Situ PCR

5 min 2 min per bath −60 min 30−

−6 h 2− 4°°C 3 × 10 min
−196°°C −160°°C −80°°C or −20°°C

❷ Sections The sections are made by cryomicrotomy: −10 µ m • Thickness 7− • Placed on Perkin–Elmer or silanated slides. −20°°C • Stored in hermetically sealed boxes with a desiccant (e.g., silicagel).

10.1.2.2.3 FIXED PARAFFIN-EMBEDDED TISSUE ❶ Preparation of tissue a. Fix: −6 h • 4% paraformaldehyde 2− 4°°C b. Rinse: • 0.1 M phosphate buffer 2 × 15 min c. Dehydrate: −2 h • Alcohol 70°, 95°, 100° 1− per bath

➫ Storage is possible at −20°C.

➫ Depending on the size of the sample

➫ Cryoprotection ➫ The samples must not float to the surface of the solution, but rather sink to the bottom of the container. ➫ Freezing can also be carried out by contact with dry ice. ➫ This method is easier to reproduce. ➫ If possible ➫ For a few days, if the samples are mounted in O.C.T

➫ See Appendix A3. ➫ Several months ➫ Note: Nonfixed tissue sections must be fixed with 4% paraformaldehyde before any pretreatment is carried out.

➫ Depending on the size of the sample ➫ Or room temperature

283

Typical Protocols d. Impregnate: • Xylene or methyl cyclohexane • A solvent mixture, paraffin (1:1 v/v) • Paraffin

−2 h 1− −2 h 1− 56°°C −4 h 2 × 1− 56°°C

e. Embed in paraffin. ❷ Sections The sections are made with a microtome. −10 µm • Thickness 7− • Placed on Perkin-Elmer slides

• Placed on silanated slides ❸ Storage Storage in hermetically sealed boxes with a desiccant (e.g., silica gel)

10.1.2.3

rt

10.1.2.3.1 CELL CULTURES AND FROZEN SECTIONS ❶ The return of the slides to 60 min room temperature rt ❷ Rehydration • Ethanol 100°, 95°, 70° 2 min per bath • 9‰ NaCl 5 min ❸ Proteolytic digestion a. Incubate the slides: • Tris–HCl/CaCl2 5 min −2 µg/ml • Tris–HCl/CaCl2; 1− proteinase K 15 min 37°°C

c. Rinse: • Sterile Tris–HCl/CaCl2 d. Dehydrate: • Ethanol 95°, 100° • Dry the sections under a hood. 284

➫ Toxic vapors

➫ Each slide can take two or three sections, which makes it possible to carry out different controls in the same experimental conditions (see Figure 5.4). ➫ See Appendix A3. ➫ Room temperature ➫ Several years ➫ For storage purposes, samples should preferably be embedded in paraffin blocks. ➫ See Chapter 3.

Pretreatments

b. Deactivate the enzyme.

➫ Or the equivalent

2 min 95°°C

➫ Condensation due to the warming of the slides should have disappeared.

➫ Proteinase K must be added extemporaneously to the buffer at 37°C. Note: This digestion may cause the destruction of cells and produce background noise or a false-negative result due to the diffusion of the amplified product. ➫ Perform on a heating block. ➫ The proteolytic digestion can also be stopped by a Tris–HCl/CaCl2 bath, with the addition of 2 mg/ml glycine.

2 × 5 min 2 min per bath −2 h 1−

➫ Pretreated slides can be stored at −20°C.

10.1 ❹ Postfixation a. Immediately after the proteolytic digestion step, the samples can be postfixed to ensure the best adhesion of the section to the slide, and to stabilize the more fragile structures. • 4% paraformaldehyde 5 min • 0.1 M phosphate buffer 2 min • 9‰ NaCl 2 min b. Dehydrate: • Ethanol 95°, 100° 2 min per bath −2 h • Dry the slides under a hood. 1− 10.1.2.3.2 PARAFFIN-EMBEDDED FIXED TISSUE ❶ Dewaxing • Xylene 2 × 15 min • Alcohol 100° 10 min • Alcohol 100° 5 min • Alcohol 95° 5 min • Alcohol 70° 5 min • 9‰ NaCl 5 min ❷ Proteolytic digestion a. Incubate the slides: • Tris–HCl/CaCl2 5 min −5 µg/ml • Proteinase K in 2− Tris–HCl/CaCl2 15 min 37°°C

b. Deactivate the enzyme.

c. Rinse: • Tris–HCl/CaCl2 d. Dehydrate: • Ethanol 95°, 100° e. Dry the slides under a ventilated hood. 10.1.2.4

2 min 95°°C

2 × 5 min

2 min per bath −2 h 1−

In situ amplification

❶ Reaction mixture for in situ amplification using labeled dNTP a. In a microtube placed in ice, prepare a reaction mixture containing:

Direct in Situ PCR

➫ An optional step

➫ Pretreated slides can be stored at −20°C.

➫ Or the equivalent

➫ Proteinase K must be added extemporaneously to the buffer at 37°C. ➫ This concentration depends on the type of tissue. ➫ The risk of morphological destruction is much smaller for paraffin-embedded tissue. ➫ Perform on a heating block. ➫ The proteolytic digestion can also be stopped at room temperature by a bath of Tris–HCl/ CaCl2, with the addition of 2 mg/ml of glycine. ➫ Postfixation is not necessary for paraffinembedded tissue. ➫ Pretreated slides can be stored at −20°C. ➫ See Chapter 5. ➫ The labels are essentially antigenic. ➫ During this amplification, nucleotides labeled with biotin are incorporated directly during the synthesis of the PCR products. 285

Typical Protocols • • • •

10X PCR buffer 50 mM MgCl2 10 mM dTTP, dCTP, dGTP 0.4 mM biotin-14-dATP

• 10 mM dATP • 10 mM sense primer

10 µ l 3 µl 2 µl each 25 µ l 1 µl 3 µl

• 10 µM anti-sense primer 3 µl • 5 U/µl Taq DNA polymerase 2.3 µ l • DEPC water 46.7 µ l b. Place the reaction mixture on the sections. c. Cover the sections with ampli cover disks and cover clips (Perkin-Elmer) or Easyseal (Hybaid). d. Place the slides in a thermocycler. ❷ Reaction mixture for in situ amplification using labeled primers

a. In a microtube placed in ice, prepare the reaction mixture, with: • 10X PCR buffer 10 µ l • 50 mM MgCl2 3 µl • dNTP 2 µl each • 10 µM sense primer 10 µ l • 10 µM anti-sense primer 10 µ l • 5 U/µl Taq DNA polymerase 2.3 µ l • DEPC water 56.7 µ l b. Place the reaction mixture on the sections.

c. Cover the sections with ampli cover disks and cover clips (Perkin-Elmer), Easyseal (Hybaid); or use another method. d. Place the slides in a thermocycler. ❸ Amplification cycles a. Denature: • Denaturing DNA 5 min 95°°C b. Preform the following cycles (20 cycles):

286

➫ Final concentration: 1X ➫ Final concentration: 1.5 mM ➫ Final concentration: 0.2 mM ➫ Final concentration: 0.1 mM ➫ Other labeled nucleotides (e.g., dUTPdigoxygenin, or dUTP-fluorescein) can be used. ➫ Final concentration: 0.1 mM ➫ Final concentration: 0.3 µM ➫ Labeled primers can be used (see Section 5.3.2.7). ➫ Final concentration: 0.3 µM ➫ Final concentration: 0.1 U/µl ➫ To a final volume of 100 µ l ➫ Volume: 30 to 50 µl, depending on the size of the sections ➫ To avoid evaporation during the hightemperature cycles ➫ During this amplification, the primers that were labeled during their synthesis by the coupling of antigenic molecules hybridize to one of the two strands of the denatured DNA and neosynthesize a complementary strand by taking it as a matrix. ➫ Final concentration: 1X ➫ Final concentration: 1.5 mM ➫ Final concentration: 0.2 mM ➫ 10 mM dTTP, dATP, dCTP, dGTP ➫ Final concentration: 1 µM ➫ Final concentration: 1 µM ➫ Final concentration: 0.1 U/µl ➫ To a final volume of 100 µ l ➫ Volume: 30 to 50 µl. The slides are placed on a heating block at 95°C, or the special Perkin-Elmer apparatus. ➫ To avoid evaporation during the hightemperature cycles

➫ To eliminate protease ➫ Necessary to optimize the number of cycles with regard to the DNA in question, the tissue, and the primers

10.1 • Denaturing • Hybridization

• Extension c. Perform final extension. d. Stop the reaction.

1 min 95°°C 90 s 57°°C 90 s 74°°C 2 min 74°°C 10 s 30°°C

e. Wash: • 0.1 M Tris–HCl buffer ❹ Postfixation • Cold ethanol 100° ❺ Rehydration • Alcohol 95°, 70°

10.1.2.5

2 × 5 min 10 min −20°°C

Direct in Situ PCR

➫ Necessary to optimize the hybridization temperature with regard to the characteristics of the primers chosen

➫ Possible to place the slides on hold if the thermocycler has been programmed at 4°C ➫ After removing the clips, cover disks, or other ➫ To eliminate diffused products ➫ Fixation of the amplified products

5 min per bath

Detection

❶ Direct detection by conjugated streptavidin a. Incubate the slides in 5 min Tris–HCl/NaCl buffer, with the additon of a blocking agent. b. Incubate: • Streptavidin conjugated to 1h alkaline phosphatase diluted 37°°C to 1:500 in Tris–HCl/NaCl buffer, with the addition of 1% ovalbumin c. Rinse: • Tris–HCl/NaCl buffer 3 × 10 min ❷ Direct detection by a conjugated antibody directed against the antigen in question a. Incubate the slides in 5 min Tris–HCl/NaCl buffer added to a blocking agent. b. Incubate: • Streptavidin conjugated 1h to alkaline phosphatase or rt peroxidase diluted to 1:100 to 1:500 in the blocking buffer, or • Anti-digoxygenin conjugated 1h to alkaline phosphatase or rt peroxidase diluted to 1:100 to 1:500 in the blocking buffer, or

➫ In a moisture chamber ➫ Possible to use streptavidin conjugated to peroxidase, but better results seem to be obtained with the alkaline–phosphatase coupling

➫ Depending on the label used

287

Typical Protocols • Anti-fluorescein, conjugated to 1h alkaline phosphatase or peroxidase rt diluted to 1:100 to 1:500 in the blocking buffer c. Rinse: • Tris–HCl/NaCl buffer 3 × 10 min ❸ Detection a. Incubate: • Tris–HCl/NaCl/MgCl2 5 min buffer, pH 9.6 b. Prepare the substrate: • NBT • BCIP • Tris–HCl/NaCl/MgCl2 buffer, pH 9.6

30 µl 40 µl 10 ml

−2 h c. Incubate until a signal 10 min− is obtained. rt d. Stop the reaction by washing 5 min in distilled water. ❹ Mounting the slides • In an aqueous medium if the substrate is NBT/BCIP or Fast Red 10.1.2.6

➫ DAB for detection with streptavidin or an IgG conjugated to peroxidase ➫ Final concentration: 0.3 mg/ml ➫ Final concentration: 0.2 mg/ml ➫ To inhibit endogenous phosphatase in some types of tissue, necessary to add levamisol (1 mM) to this preparation ➫ In the same way, possible to use hydrogen peroxide (H2O2) to inhibit endogenous peroxidase. ➫ Detection carried out in darkness under a microscope

➫ Aquamount or Glycergel ➫ See Appendix B8.1.

Controls

❶ Positive controls • Tissue or cells containing the DNA being sought ❷ Negative controls • Tissue or cells that are known not to contain the DNA being sought ❸ Reaction controls • Omission of Taq DNA polymerase • Omission of dNTP, whether labeled or not • Omission of primers, whether labeled or not ❹ Detection-reaction controls • Omission of the conjugate

288

➫ See Appendix B3.7.6.

➫ The only way to find out if the reaction has taken place normally in the case of negative results ➫ To confirm a specific amplification ➫ If the amplification step is omitted, no positive result ➫ Confirmation of the immunoenzymatic reaction ➫ No amplification step ➫ No detection step ➫ Revelation of endogenous enzymatic activity

10.2

10.2

INDIRECT IN SITU PCR

Indirect in situ PCR, like direct in situ PCR, is essentially used to detect viral DNA or to identify foreign genes in cultured cells, frozen tissue sections, or fixed, paraffin-embedded tissue sections. Hybridization of the amplified product with the appropriate probes gives a further degree of specificity. The sample preparation and pretreatment steps are exactly the same as for direct PCR.

Indirect in Situ PCR

➫ See Chapter 11, Figures 11.1 through 11.3.

➫ Gloves must be worn. All the products must be RNase-free, the solutions must be prepared in DEPC water (see Appendix B1.2), and the equipment must be sterilized (see Appendix A2). ➫ See Chapters 2 and 3.

10.2.1 Diagram of the Different Steps

289

Typical Protocols

10.2.2 Typical Protocols 10.2.2.1

290

Solutions

Ammonium acetate Anti-digoxygenin conjugated to alkaline phosphatase or peroxidase Anti-fluorescein conjugated to alkaline phosphatase or peroxidase BCIP Blocking agent 10 mM dTTP, dCTP, dGTP dATP 10 mg/ml salmon sperm DNA Ethanol 100° (−20°C) Ethanol 70°, 95°, 100° Isopentane Liquid nitrogen 50 mM MgCl2 9‰ NaCl NBT Paraffin 4% paraformaldehyde 10X PCR buffer 0.1 M phosphate buffer 10 µM anti-sense primer 10 µM sense primer Anti-sense probe Sense probe Proteinase K 10 mg/ml tRNA 20X SSC 0.1 M Tris–HCl buffer Tris–HCl/CaCl2 buffer Tris–HCl/NaCl buffer Tris–HCl/NaCl/MgCl2 buffer 5 U/µl Taq DNA polymerase DEPC water Sterile water Xylene or methyl cyclohexane

➫ See Appendix B2.2.

10.2.2.2

Preparation of samples

➫ See Section 10.1.2.2.

10.2.2.3

Pretreatments

➫ See Section 10.1.2.3.

➫ ➫ ➫ ➫

See Appendix B6.2.2.1. See Appendix B6.2.1. dNTP See Appendix B2.8.

➫ See Appendix B2.12. ➫ See Appendix B2.19. ➫ See Appendix B6.2.2.1. ➫ See Appendix B4.3.2. ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B3.4.1. See Section 5.3.2. See Section 5.3.2. See Section 6.3. See Section 6.3. See Appendix B2.14.1. See Appendix B2.15. See Appendix B3.5. See Appendix B3.7.1. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.4. Supplied with the enzyme See Appendix B1.2. See Appendix B1.1.

10.2 10.2.2.4

In situ amplification

❶ Reaction mixture a. In a microtube placed in ice, prepare the reaction mixture: • 10X PCR buffer 10 µ l • 50 mM MgCl2 3 µl • 10 mM dNTP 2 µl each • 10 µM sense primer 3 µl • 10 µM anti-sense primer 3 µl • 5 U/µl Taq DNA polymerase 2.3 µ l • Sterile water 70.7 µ l b. Place the reaction mixture on the sections.

c. Cover the sections with ampli cover disks and clips (Perkin-Elmer), or Easyseal (Hybaid). d. Place the slides in a thermocycler. ❷ Amplification cycles a. Denature: • Denaturing the DNA 5 min 95°°C b. Perform the following cycles: • Denaturing 1 min 95°°C • Hybridization 90 s 57°°C

• Extension c. Perform final extension. d. Stop the reaction.

90 s 74°°C 3 min 74°°C 10 s 30°°C

e. Wash: • Tris–HCl buffer ❸ Fixation • Cold ethanol 100° (−20°C) ❹ Drying • Under a ventilated hood 10.2.2.5 10.2.2.5.1

Indirect in Situ PCR

2 × 5 min 10 min

➫ Final concentration: 1X ➫ Final concentration: 1.5 mM ➫ Final concentration: 0.2 mM ➫ Final concentration: 0.3 µM ➫ Final concentration: 0.3 µM ➫ Final concentration: 0.1 U/µl ➫ To a final volume of 100 µ l ➫ Volume: 50 µl ➫ On a heating block at 95°C, or in the special Perkin-Elmer apparatus ➫ To avoid evaporation during the hightemperature cycles

➫ 20 cycles

➫ The hybridization temperature must be optimized with regard to the characteristics of the primers chosen.

➫ The slides can be put on hold after the thermocycler has been programmed at 4°C. ➫ Remove the clips and ampli cover disks, or other. ➫ Elimination of the diffused products ➫ Fixation of the amplified products

30 min–1 h

Hybridization LABELING PROBES BY EXTENSION 3′ END

OF THE

➫ Hybridization after amplification is generally carried out with oligonucleotidic probes, and is thus the most commonly used type of labeling.

291

Typical Protocols ❶ Reaction mixture a. In a sterile microtube, prepare the reaction mixture: • 100 pmol/µl of oligonucleotides 1 µl • A 5X buffer specific to terminal 4 µl transferase (TdT) • 25 mM CoCl2 4 µl • 25 mmol Dig-11-dUTP 1 µl

• Sterile water • 25 U/µl terminal deoxy transferase

b. Incubate in a water bath.

5 µl 1 µl

292

➫ Final concentration: 5 mM ➫ Final concentration: 1.25 mmol ➫ Or another labeled dUTP (see Section 5.3.1.3) ➫ To a final volume of 20 µ l ➫ Final concentration: 1.25 U/µl ➫ This enzyme is delicate, and must not be heated or vortexed, but shaken gently and, if necessary, centrifuged rapidly.

1h 37°°C

c. Stop the action of the enzyme in ice. ❷ Precipitation a. Precipitate the probe by adding: • tRNA 2 µl • Cold absolute ethanol 66 µ l • Ammonium acetate 9 µl b. Store. Overnight −20°°C c. Centrifuge. 30 min 14,000 g 4°°C d. Remove the supernatant. e. Dry the pellet in a vacuum jar or a Speedvac. −20°°C ❸ Storage

10.2.2.5.2 HYBRIDIZATION ❶ Reaction medium a. In a sterile microtube placed in ice, the reaction mixture: • 20X SSC • Deionized formamide • 50X Denhardt’s solution • 10 mg/ml tRNA • 10 mg/ml salmon sperm DNA • Sense probe (1.25 pmol/µl), labeled with digoxygenin • Anti-sense probe (1.25 pmol/µl), labeled with digoxygenin • Sterile water

➫ Final concentration: 5 pmol ➫ Final concentration: 1X

➫ Store at −20°C. ➫ See Appendix B2.2. ➫ Orient the tube in such a way to best observe the position of the centrifugation pellet. ➫ Eliminate all trace of alcohol. ➫ The probes are stored dry or solubilized in sterile distilled water.

prepare 100 µ l 250 µ l 10 µ l 12.5 µ l 12.5 µ l 8 µl 8 µl 99 µ l

➫ ➫ ➫ ➫ ➫ ➫

Final concentration: 4X Final concentration: 50% Final concentration: 1X Final concentration: 250 µg/ml Final concentration: 250 µg/ml Final concentration: 20 pmol/ml

➫ Final concentration: 20 pmol/ml ➫ To a final volume of 500 µ l

10.2 b. Place the reaction mixture on the sections. c. Place sterile cover slides on the sections. d. Denature. 5 min 96°°C e. Cool immediately on ice. 5 min f. Incubate.

Overnight 40°°C

❷ Washing a. Remove the cover slides in a 4X SSC bath. b. Rinse: • 2X SSC 2 × 30 min • 0.5X SSC 30 min 10.2.2.6

Indirect in Situ PCR

➫ Volume: 30 to 50 µl ➫ To avoid evaporation ➫ On a heating block ➫ To stabilize the DNA in the form of single strands ➫ In a moisture chamber

➫ At room temperature ➫ At room temperature

Revelation

❶ Indirect detection by a conjugated antibody directed against the antigenic molecule in question a. Incubate the slides in Tris–HCl/ 5 min NaCl buffer, with the addition of a blocking agent. b. Incubate with: • Anti-biotin conjugated to alkaline 1h phosphatase or peroxidase rt • Anti-digoxygenin conjugated to 1h alkaline phosphatase or peroxidase rt • Anti-fluorescein conjugated to 1h alkaline phosphatase or peroxidase rt c. Rinse: • Tris–HCl/NaCl buffer 3 × 10 min ❷ Detection a. Incubate: • Tris–HCl/NaCl/MgCl2 buffer, 5 min pH 9.6 b. Prepare the substrate: • NBT • BCIP • Tris–HCl/NaCl/MgCl2 buffer, pH 9.6

c. Incubate until a signal is obtained. d. Stop the reaction by washing in distilled water.

30 µ l 40 µ l 10 ml

10 min–2 h rt 5 min

➫ Depending on the label ➫ Diluted to 1:100 to 1:500 in the blocking buffer ➫ Diluted to 1:100 to 1:500 in the blocking buffer ➫ Diluted to 1:100 to 1:500 in the blocking buffer

➫ See Appendix B3.7.6 ➫ DAB in the case of detection with a streptavidin or an antibody conjugated to peroxidase ➫ Final concentration: 0.3 mg/ml ➫ Final concentration: 0.2 mg/ml ➫ To inhibit the endogenous phosphatases that are found in certain types of tissue, necessary to add levamisol (1 mM) to this preparation ➫ Likewise, hydrogen peroxide (H2O2) used to inhibit endogenous peroxidases ➫ Detection carried out in darkness under a microscope

293

Typical Protocols ❸ Mounting the slides • Ιn an aqueous medium, if the substrate is NBT/BCIP or Fast Red • Ιn a synthetic medium, after dehydration in alcohol and dipping in solvents, if the substrate is DAB 10.2.2.7

Controls

❶ Positive controls • Tissue or cells containing the DNA being sought ❷ Negative controls • Tissue or cells that are known not to contain the DNA being sought ❸ Reaction controls • Omission of Taq DNA polymerase • Omission of primers • Omission of the labeled dNTP ❹ Detection controls • Omission of probes • Omission of antibodies

10.3

➫ Aquamount or Glycergel (see Appendix B8.1) ➫ Entellan or Eukitt (see Appendix B8.2)

➫ See Chapter 9. ➫ The only way to find out if the reaction has taken place normally in the case of negative results ➫ To confirm a specific amplification ➫ If the amplification step omitted, no positive result ➫ No amplification step ➫ Confirmation of the immunoenzymatic reaction ➫ Negative result ➫ Absence of phosphatases or endogenous peroxidases

INDIRECT IN SITU RT-PCR ➫ See Chapter 11, Figures 11.4 through 11.10.

This is the only way to visualize mRNA that is not strongly expressed in a particular type of cell. Thus, the detection of a weakly expressed gene, an abnormal gene, or an RNA virus is a potential application of in situ RT-PCR. The steps involved in preparing the samples, and the pretreatments, are exactly the same as for direct PCR.

294

➫ Gloves must be worn. All the products must be RNase-free, the solutions must be prepared in DEPC water (see Appendix B1.2), and the equipment must be sterilized (see Appendix A2). ➫ In the case of RNA, which breaks down very easily, these conditions must be even more strictly respected. ➫ See Sections 10.1.2.2 and 10.1.2.3.

10.3

Indirect in Situ RT-PCR

10.3.1 Diagram of the Different Steps

10.3.2 Typical Protocols 10.3.2.1

Solutions

Anti-biotin conjugated to alkaline phosphatase or peroxidase Anti-digoxygenin conjugated to alkaline phosphatase or peroxidase Anti-fluorescein, conjugated to alkaline phosphatase or peroxidase BCIP Blocking agent 2 mM CaCl2

➫ See Appendix B6.2.2.1. ➫ See Appendix B6.2.1. ➫ See Appendix B2.3.2. 295

Typical Protocols Diaminobenzidine (DAB) 10 mg/ml salmon sperm DNA DNase 0.1 M DTT 10 mM dTTP, dCTP, dGTP, dATP Ethanol 100° (−20°C) Ethanol 70°, 95°, 100° Deionized formamide 50X Denhardt’s solution Isopentane Liquid nitrogen 6 mM; 50 mM MgCl2 9‰ NaCl NBT Paraffin 4% paraformaldehyde 10X PCR buffer 0.1 M phosphate buffer Proteinase K 10 µM anti-sense primer 10 µM sense primer Anti-sense probe Sense probe 200 U/µl reverse transcriptase 10 mg/ml tRNA  40 U/µl RNasin 5X RT buffer 20X SSC Streptavidin conjugated to alkaline phosphatase 5 U/µl Taq DNA polymerase Tris–HCl buffer Tris–HCl/CaCl2 buffer Tris–HCl/NaCl buffer Tris–HCl/NaCl/MgCl2 buffer DEPC water Sterile water Xylene or methyl cyclohexane

➫ ➫ ➫ ➫ ➫

10.3.2.2

Preparation of samples

➫ See Section 10.1.2.2.

10.3.2.3

Pretreatments

➫ See Section 10.1.2.3.

10.3.2.4

DNase treatment

➫ Optional step if the primers, and especially the probes, are specific for the sequence to be amplified. ➫ The DNase must be of RNase-free quality.

a. Prepare the DNase solution: • 40 mM Tris–HCl, pH 7.4 • 6 mM MgCl2 • 2 mM CaCl2 296

See Appendix B6.2.2. See Appendix B2.8. See Appendix B2.9. See Appendix B2.7. dNTP

➫ See Appendix B2.4. ➫ See Appendix B2.5. ➫ See Appendix B2.12. ➫ See Appendix B2.19. ➫ See Appendix B6.2.2.1. ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B4.3.2. Supplied with the enzyme See Appendix B3.4.1. See Appendix B2.14.1. See Section 5.3.2. See Section 5.3.2. See Section 6.3. See Section 6.3.

➫ See Appendix B2.15. ➫ Supplied with the enzyme ➫ See Appendix B3.5. ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B3.7.1. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.4. See Appendix B1.2. See Appendix B1.1.

10.3 • 1 to 100 U/ml of DNase in the final volume

b. Cover the sections with 20 to 30 µl of this solution. c. Incubate in a moisture chamber. 1–12 h 37°°C d. Rinse the sections in the DNase dilution buffer. e. Deactivate the enzyme. 2 min 95°°C f. Rinse the slides in DEPC water. 10.3.2.5

➫ For cells and tissue that are particularly rich in ribonuclease, 1000 U/ml RNasin and 1 mM dithiothreitol (DTT) should be added to the DNase solution. ➫ Time depends on the concentration.

➫ On a heating block

Reverse transcription

❶ Reaction mixture a. In a sterile microtube, prepare the reaction mixture: • 5X RT buffer 20 µ l • 0.1 M DTT 10 µ l • 10 mM dNTPs 5 µl • 40 U/µl RNasin 2.5 µ l • 10 µM anti-sense primer 10 µ l • DEPC water 47.5 µ l • 200 U/µl reverse transcriptase 5 µl b. Place the reaction mixture on the sections. c. Cover the sections with ampli cover disks and cover clips (Perkin-Elmer), Easyseal (Hybaid), or simply sealed cover slides. d. Place the slides 60 min in the thermocycler. 40°°C e. Deactivate the enzyme. 2 min 94°°C ❷ Washing • 0.1 M phosphate buffer 5 min • 9‰ NaCl 2 min ❸ Dehydration • Ethanol 95°, 100°

➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫

Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/µl Final concentration: 1 µM To a final volume of 100 µ l Final concentration: 10 U/µl Volume: 30 to 50 µl To avoid evaporation See Section 4.4.2

➫ The temperature at which reverse transcriptase activity is optimal ➫ The temperature, at which the enzyme is destroyed ➫ To avoid phosphate precipitation (whitish streaks)

2 min per bath

❹ Drying • Under a ventilated hood

10.3.2.6

Indirect in Situ RT-PCR

➫ Traces of alcohol could precipitate the polymerase.

In situ amplification

❶ Reaction mixture In a sterile microtube placed in ice, prepare the reaction mixture: • 10X PCR buffer 10 µ l

➫ Final concentration: 1X 297

Typical Protocols • 25 mM MgCl2 • Mixture of 10 mM dNTP • 10 µM sense primer • 10 µM anti-sense primer • DEPC water ❷ The hot start

6 µl 5 µl 10 µ l 10 µ l 55 µ l

5 min 82°°C b. Add 5 U/µl Taq DNA polymerase. 4 µl c. Place the reaction mixture on the sections.

➫ Final concentration: 1.5 mM ➫ Final concentration: 0.2 mM ➫ Final concentration: 1 µM ➫ Final concentration: 1 µM ➫ To a final volume of 96 µ l ➫ The specificity of primer matching must be ensured. There is a high risk of nonspecific hybridization at low temperature.

a. Incubate the mixture.

d. Cover the sections with the ampli cover disks and clips (Perkin-Elmer), Easyseal (Hybaid), or simply sealed cover slides. e. Place the slides in a thermocycler. ❸ Amplification cycles a. Program 25 amplification cycles: • Denaturing 1 min 94°°C • Hybridization 90 s 45°°C • Extension b. Perform final extension. c. Stop the reaction.

90 s 72°°C 5 min 72°°C 10 s 30°°C

d. Wash: • 0.1 M phosphate buffer ❹ Postfixation • 4% paraformaldehyde • 0.1 M phosphate buffer • 9‰ NaCl ❺ Dehydration • Ethanol 95°, 100° ❻ Drying • Under a ventilated hood 10.3.2.7

2 × 5 min 15 min 5 min 2 min

➫ The hybridization temperature must be optimized according to the characteristics of the primers.

➫ The slides can be stored at 4°C in the thermocycler. ➫ Remove the clips and the ampli-cover disks, or other. ➫ Eliminate diffused products. ➫ Fix the amplified products.

2 min per bath 30 min–1 h

Hybridization

❶ Reaction mixture a. In a sterile microtube placed in ice, prepare the reaction mixture: • 20X SSC 100 µ l 298

➫ Final concentration: 0.2 U/µl ➫ Volume: 50 µl. The sections are placed on a heating block at 95°C, or the special PerkinElmer apparatus. ➫ To avoid evaporation during the hightemperature cycles

➫ Final concentration: 4X

10.3 Deionized formamide 250 µ l 50X Denhardt’s solution 10 µ l 10 mg/ml tRNA 12.5 µ l 10 mg/ml salmon sperm DNA 12.5 µ l Sense probe (1.25 pmol/µl) 8 µl labeled with digoxygenin • Anti-sense probe (1.25 pmol/µl) 8 µl labeled with digoxygenin • Sterile water 99 µ l b. Place the reaction mixture on the slides. c. Place sterile coverslips on the sections. d. Denature. 5 min 96°°C e. Cool immediately on ice. 5 min • • • • •

f. Incubate.

Overnight 40°°C

❷ Washing a. Detach the coverslips in a 4X SSC bath. b. Rinse: • 2X SSC 2 × 30 min • 0.5X SSC 30 min 10.3.2.8

➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫

Indirect in Situ RT-PCR

Final concentration: 50% Final concentration: 1X Final concentration: 250 µg/ml Final concentration: 250 µg/ml Final concentration: 20 pmol/ml Or another label (e.g., biotin fluorescein) Final concentration: 20 pmol/ml Or another label (e.g., biotin fluorescein) To a final volume of 500 µ l Volume: 30 to 50 µl To avoid evaporation On a heating block

➫ To stabilize the DNA in the form of single strands ➫ In a moisture chamber containing 5X SSC

➫ At room temperature ➫ At room temperature

Revelation

❶ Direct detection by a conjugated antibody directed against the antigenic molecule being used a. Incubate the slides in Tris–HCl/ 5 min NaCl, added to a blocking agent. b. Incubate with: • Anti-digoxygenin conjugated to 1h alkaline phosphatase or peroxidase rt

c. Rinse: • Tris–HCl/NaCl buffer 3 × 10 min ❷ Phosphatase alkaline detection a. Incubate: • Tris–HCl/NaCl/MgCl2 buffer, 5 min pH 9.6 b. Prepare the substrate extemporaneously:

• NBT • BCIP • Tris–HCl/NaCl/MgCl2 buffer, pH 9.6

30 µ l 40 µ l 10 ml

➫ Ovalbumin, goat serum ➫ Depending on the label ➫ Diluted to 1:100 to 1:500 in the blocking buffer ➫ Or another system, depending on the label (e.g., anti-biotin or anti-fluorescein)

➫ DAB in the case of detection with an antibody conjugated with peroxidase (see Chapter B6.2.2.2) ➫ Final concentration: 0.3 mg/ml ➫ Final concentration: 0.2 mg/ml ➫ To inhibit the phosphatases that are endogenous to certain types of tissue, necessary to add levamisol (1 mM) to this preparation ➫ In the same way, hydrogen peroxide (H2O2) used to inhibit endogenous peroxidases 299

Typical Protocols c. Incubate until a signal is 10 min–2 h obtained. rt d. Stop the reaction by washing 5 min in Tris–HCl buffer. ❸ Mounting the sections • In an aqueous medium if the substrate is NBT/BCIP or Fast Red • In a synthetic medium after dehydration in alcohol, and baths of solvent if the substrate is DAB 10.3.2.9

Controls

❶ Positive controls • Tissue or cells containing the mRNA being sought ❷ Negative controls • Tissue or cells known not to contain the mRNA being sought ❸ Reaction controls • Omission of reverse transcriptase • Omission of Taq DNA polymerase • Omission of primers • Omission of labeled dNTP ❹ Detection controls • Omission of probes

• Omission of antibodies

10.4

➫ Aquamount or Glycergel (see Appendix B8.1) ➫ Entellan, Eukitt (see Appendix B8.2)

➫ See Chapter 9. ➫ The only way to find out if the reaction has taken place normally in the case of negative results ➫ To confirm a specific amplification

➫ If the amplification step omitted, no positive result ➫ No amplification step ➫ Confirmation of the immunoenzymatic reaction ➫ Possible that a weak signal may persist even though there is no amplification step; which results from the hybridization of cDNA synthesized during the RT step ➫ Negative results; the demonstration of endogenous enzymatic activity

DIRECT IN SITU RT-PCR SPECIAL CASE: CELL SUSPENSION

This protocol is applicable to all suspended cells, whether derived from cultures, biological fluids, or even the mechanical or enzymatic dissociation of tissue. The isolation protocols are different in each case, and we would simply recall

300

➫ Detection carried out in darkness under a microscope

➫ Gloves must be worn. All the products must be RNase-free, solutions must be prepared in DEPC water (see Appendix B1.2), and the equipment must be sterilized (see Appendix A.2).

10.4 that the integrity of the cells must be conserved, as RT and PCR reactions take place in the cytoplasm. Here we will give the fixation and permeabilization procedures that must be carried out before RT-PCR.

Direct in Situ RT-PCR

➫ In the case of RNA, which breaks down very easily, these conditions must be even more strictly respected.

10.4.1 Diagram of the Different Steps

Direct in situ RT-PCR using flourescent primers

301

Typical Protocols Direct in situ RT-PCR using biotinylated primers

10.4.2 Typical Protocol 10.4.2.1

Solutions

Anti-biotin conjugated to alkaline phosphatase or peroxidase Anti-digoxygenin conjugated to alkaline phosphatase or peroxidase Anti-fluorescein conjugated to alkaline phosphatase or peroxidase BCIP 302

➫ See Appendix B6.2.2.1

10.4 0.4 mM biotin-14-dATP Blocking agent 2 mM CaCl2 50X Denhardt’s solution Diaminobenzidine (DAB) 10 mg/ml salmon sperm DNA 0.1 M DTT 10 mM dTTP, dCTP, dGTP dATP dUTP conjugated to digoxygenin dUTP conjugated to fluorescein Ethanol 100° (–20°C) Ethanol 70°, 95°, 100° 10% formol Hémalun’s solution Igepal CA-630 Isopentane 50 mM; 6 mM MgCl2 9‰ NaCl NBT Paraffin 4% paraformaldehyde PBS PBS-glycerol 0.1 M phosphate buffer 10X PCR buffer 10 µM anti-sense primer 10 µM sense primer Anti-sense probe Sense probe Proteinase K 20X SSC 200 U/µl reverse transcriptase 10 mg/ml tRNA 40 U/µl RNasin 5X RT buffer Streptavidin conjugated to alkaline phosphatase 5 U/µl Taq DNA polymerase 40 mM; 100 mM Tris–HCl buffer Tris–HCl/CaCl2 buffer Tris–HCl/NaCl buffer Tris–HCl/NaCl/MgCl2 buffer DEPC water Sterile water Xylene or methyl cyclohexane 10.4.2.2

➫ ➫ ➫ ➫ ➫ ➫

Direct in Situ RT-PCR

See Appendix B6.2.1. See Appendix B2.3. See Appendix B2.5. See Appendix B6.2.2.2. See Appendix B2.8. See Appendix B2.7.

➫ See Appendix B4.1. ➫ See Appendix B7.1.3. ➫ See Appendix B2.12. ➫ See Appendix B2.19. ➫ See Appendix B6.2.2.1. ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B4.3.2. See Appendix B4.3.4.3. See Appendix B8.1. See Appendix B3.4.1. Supplied with the enzyme See Section 5.3.2. See Section 5.3.2. See Section 6.3. See Section 6.3. See Appendix B2.14.1. See Appendix B3.5.

➫ See Appendix B2.15. ➫ Supplied with the enzyme ➫ ➫ ➫ ➫ ➫

See Appendix B3.7.1. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.4. See Appendix B1.2.

Fixation

The cells are rinsed in PBS before being centrifuged and separated into aliquots of a minimum 7 of 10 cells in sterile microtubes.

➫ The minimum number of cells is important, since subsequent washes and operations lead to the loss of a large number of cells. 303

Typical Protocols a. Centrifuge.

3 min 1500 g b. Remove the supernatant and put the suspended pellet in 1 ml of 4% paraformaldehyde or 10% formol, and 0.15 M NaCl. c. Incubate in ice, shaking frequently. 1h d. Centrifuge. 3 min 1500 g e. Suspend the pellet in PBS. 4°°C f. Centrifuge. 3 min 1500 g Perform this washing step three times. 10.4.2.3

➫ Use a “Pasteur”-type pipette to suspend the pellet. ➫ See Appendix B2.19. ➫ It is important to use a refrigerated centrifuge.

Pretreatments

a. Suspend the pellet in Igepal CA-630. 0.5%

b. Incubate, shaking frequently. c. Centrifuge. d. Suspend the pellet in PBS.

1h 0°°C 3 min 1500 g 4°°C

➫ Nonionic detergent whose chemical composition is similar to that of Nonidet P 40, which is no longer available ➫ Possible to use other detergents ➫ Melting ice ➫ Important to use a refrigerated centrifuge

Perform this washing step three times. e. After the last centrifugation, count the cells, make sure that they are not clustered, and 6 adjust the concentration to 2 × 10 cells/ml. 10.4.2.4

Reverse transcription

❶ Reaction mixture a. In a sterile microtube, prepare the reaction mixture: • 5X RT buffer 10 µ l • 0.1 M DTT 5 µl • 10 mM dNTPs 2.5 µ l • 40 U/µl RNasin 2.5 µ l • 10 µM anti-sense primer 5 µl • DEPC water 22.5 µ l b. Suspend the pretreated cell pellet in the calculated quantity of distilled water. c. Add the reaction mixture, and homogenize by careful pipetting. d. Add 200 U/µl reverse transcriptase, 2.5 µl and mix once more by careful pipetting. e. Incubate. 1h 37°°C or, 1h 42°°C 304

➫ If the cells are not to be used immediately, the aliquots can be frozen and stored at −80°C.

➫ ➫ ➫ ➫ ➫ ➫

Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/µl Final concentration: 1 µM To a final volume of 47.5 µ l

➫ Final concentration: 10 U/µl ➫ Final volume: 50 µ l ➫ If MMLV is the reverse transcriptase used ➫ If AMV is the reverse transcriptase used

10.4 f. Deactivate the enzyme. ❷ Washing a. Centrifuge.

2 min 94°°C

➫ At this temperature, the enzyme is destroyed.

3 min 1500 g

➫ Although the pellet is generally quite visible, it is a good idea always to place the tube in the same position in the centrifuge.

b. Remove all the supernatant, and suspend the pellet in 200 µl PBS. c. Centrifuge once more. 2 min 1500 g d. Remove the supernatant, and suspend the pellet in 100 µl PBS.

10.4.2.5

Direct in Situ RT-PCR

➫ The cells can be used directly in PCR. ➫ They can also be frozen and stored at −80°C in aliquots of 10 µl.

Amplification with labeled primers

10.4.2.5.1 FLUORESCENT PRIMERS For some applications, amplification can be carried out in the presence of labeled primers, notably a fluorochrome for the direct visualization of the amplified product. Although not recommended, this method makes possible the detection of very small numbers of positive cells by flow cytometry. ❶ Reaction mixture a. In a sterile microtube, prepare the reaction mixture: • 10X PCR buffer 5 µl • 25 mM MgCl2 3 µl • Mixture of 10 mM dNTP 2.5 µ l • 10 µM labeled sense primer 5 µl

• 10 µM labeled anti-sense primer 5 µl • Sterile water 27.5 µ l b. Add the reaction mixture to the cell pellet, and homogenize by careful pipetting. ❷ The hot start

a. Incubate the mixture. b. Add 5 U/µl Taq DNA polymerase, and check that the tube is closed.

5 min 82°°C 2 µl

➫ The use of two fluorochromes makes it possible to detect two different mRNA sequences. This codetection can be an advantage for a number of applications.

➫ Final concentration: 1X ➫ Final concentration: 1.5 mM ➫ Final concentration: 0.2 mM ➫ Final concentration: 1 µM ➫ Only one labeled primer can be used, but to this extent the effectiveness of the amplification will decline. ➫ Final concentration: 1 µM ➫ To a final volume of 48 µ l ➫ The specificity of primer matching must be ensured. There is a high risk of nonspecific hybridization at low temperature. ➫ Final concentration: 0.2 U/µl ➫ Final volume: 50 µ l ➫ To facilitate the washing steps, it is best not to add mineral oil.

305

Typical Protocols ❸ Amplification cycles a. Program 25 amplification cycles: • Denaturing • Hybridization

• Extension b. Perform final extension. c. Stop the reaction. ❹ Washing a. Centrifuge.

1 min 94°°C 90 s 45°°C 90 s 72°°C 5 min 72°°C 10 s 30°°C 3 min 1500 g

b. Remove all the supernatant and suspend the pellet in 200 µl PBS. c. Centrifuge once more. 2 min 1500 g d. Remove the supernatant and suspend the pellet in 100 µl PBS-glycerol (1:1 v/v). e. Place a 50 µl drop on a slide, and cover it with a cover slide. f. Remove the supernatant and suspend the pellet in 0.5 ml PBS. 10.4.2.5.2

➫ The slides can be put on hold after the thermocycler has been programmed at 4°C. ➫ If mineral oil has been used, it is important, to avoid contamination, to extract the cells from the liquid phase and suspend them in 200 µl PBS before centrifuging.

➫ For examination by fluorescence microscope or confocal microscope ➫ For flow cytometry studies

BIOTINYLATED PRIMERS

❶ Reaction mixture a. In a sterile microtube placed in ice, prepare a reaction mixture containing: • PCR 10X buffer 5 µl • 25 mM MgCl2 3 µl • 10 mM dNTP mixture 2.5 µ l • 10 µM labeled sense primer 5 µl

• 10 µM anti-sense primer 5 µl • Sterile water 27.5 µ l b. Add the reaction mixture to the cell pellet, and homogenize by careful pipetting. ❷ The hot start

306

➫ The hybridization temperature must be optimized in keeping with the characteristics of the primers used.

➫ Final concentration: 1X ➫ Final concentration: 1.5 mM ➫ Final concentration: 0.2 mM ➫ Final concentration: 1 µM ➫ Only one labeled primer can be used, but to this extent the effectiveness of the amplification will decline. ➫ Final concentration: 1 µM ➫ To a final volume of 48 µ l ➫ The specificity of primer matching must be ensured. There is a high risk of nonspecific hybridization at low temperature.

10.4 a. Incubate the mixture. b. Add 5 U/µl Taq DNA polymerase, and check that the tube is closed. ❸ Amplification cycles a. Program 25 amplification cycles: • Denaturing • Hybridization

• Extension b. Perform final extension. c. Stop the reaction. ❹ Washing a. Centrifuge

5 min 82°°C 2 µl

1 min 94°°C 90 s 45°°C

Direct in Situ RT-PCR

➫ Final concentration: 0.2 U/µl ➫ Final volume: 50 µ l ➫ To facilitate the washing steps, it is best not to add mineral oil.

➫ The hybridization temperature must be optimized in keeping with the characteristics of the primers used.

90 s 72°°C 5 min 72°°C 10 s 30°°C 3 min 1500 g

b. Remove all the supernatant and suspend the pellet in 200 µl PBS. c. Centrifuge once more. 2 min 1500 g d. Remove all the supernatant and suspend the pellet in 200 µl PBS. ❺ Peroxidase detection a. Place 50 µl of the suspension on a pretreated slide, and spread it carefully to obtain a smear. b. Leave to dry. 30 min c. Incubate in a moisture chamber 1h with 200 µl of streptavidin rt conjugated with the peroxidase diluted to 1:100 in Tris–HCl/NaCl. d. Rinse in Tris–HCl/NaCl in 5 min a tray, with light shaking. ≈5 min e. Incubate with 200 µl of DAB prepared extemporaneously. f. Rinse in Tris–HCl/NaCl in a tray, 5 min with light shaking. g. A counterstaining can be carried out 30 s by dipping the slides in Hemalun’s solution.

➫ If mineral oil has been used, it is important, to avoid contamination, to extract the cells from the liquid phase and suspend them in 200 µl PBS before centrifuging.

➫ If the cells are to adhere correctly, it is essential that the slides be pretreated (see Appendix A3). ➫ Streptavidin can be conjugated to alkaline phosphatase ➫ Anti-biotin conjugated can also be used.

➫ Keep an eye on the detection procedure.

➫ Check that this counterstaining does not interfere with the interpretation of the signal. ➫ See Appendix B7.1.3.

307

Typical Protocols h. Rinse rapidly in running water, 2 min and dehydrate in alcohol baths per bath of increasing strength, followed by a bath of methyl cyclohexane. ® i. Mount the slide in Entellan or DPX.

10.4.2.6

Controls

❶ Positive controls • Cells that contain the mRNA being sought. ❷ Negative controls • Cells that are known not to contain the mRNA being sought ❸ Reaction controls • Omission of reverse transcriptase • Omission of Taq DNA polymerase • Omission of labeled primers ❹ Diffusion control • 10 µl of the reaction mixture is removed after the amplification and subjected to electrophoresis.

10.5

➫ Observation is by conventional microscopy. The blue nuclei contrast with the brown cytoplasmic staining of the amplified cDNA.

➫ The only way to find out if the reaction has taken place normally in the case of negative results ➫ To confirm a specific amplification ➫ Negative control; only the hybridization of RNA can give rise to a signal ➫ If the amplification step omitted, no positive result should be obtained ➫ No amplification step ➫ If the amplified products have diffused, a band will be clearly visible on the gel.

INDIRECT IN SITU RT-PCR ON VEGETABLE TISSUE USING FLOATING VIBRATOME SECTIONS ➫ See Chapter 11, Figure 11.11.

For vegetable tissue the method of choice for visualizing weakly expressed mRNA is an adaptation of the in situ RT-PCR on floating vibratome sections, which can be applied to vegetable tissue with only small modifications. The difficulties concern the tissue structures. In the example given here, the use of thick, fixed tissue sections made on a vibratome ensures a high detection threshold in well-conserved tissue structure.

308

➫ Gloves must be worn. All the products must be RNase-free, the solutions must be prepared in DEPC water (see Appendix B1.2), and all equipment must be sterilized (see Appendix A2). ➫ In the case of RNA, which breaks down very easily, these conditions must be even more strictly respected.

10.5

Indirect in Situ RT-PCR on Vegetable Tissue Using Floating Vibratome Sections

10.5.1 Diagram of the Different Steps

309

Typical Protocols

10.5.2 Typical Protocol 10.5.2.1

Solutions

5% agarose Blocking agent Anti-digoxygenin conjugated to alkaline phosphatase or peroxidase 10 mg/ml tRNA 50X Denhardt’s solution 10 mg/ml salmon sperm DNA 0.1 M DTT 10 mM dTTP, dCTP, dGTP, dATP 6 mM; 50 mM MgCl2 9‰ NaCl 4% paraformaldehyde PBS 10X PCR buffer 0.1 M phosphate buffer 10 µM anti-sense primer 10 µM sense primer Anti-sense probe Sense probe Proteinase K 200 U/µl reverse transcriptase 10 mg/ml RNase A 40 U/µl RNasin 5X RT buffer 20X SSC 5 U/µl Taq DNA polymerase 0.1 M Tris–HCl buffer 40 mM Tris–HCl buffer Tris–HCl/CaCl2 buffer Tris–HCl/NaCl buffer Tris–HCl/NaCl/MgCl2 buffer DEPC water Sterile water 10.5.2.2

➫ ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B3.4.1. See Section 5.3.2. See Section 5.3.2. See Section 6.3. See Section 6.3. See Appendix B2.14.1. MMLV

➫ See Appendix B3.5. ➫ ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B3.7.1. See Appendix B3.7.1. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.4. See Appendix B1.2. See Appendix B1.1.

Fixation

a. Fix the sample in 4% 12 h paraformaldehyde in 100 mM phosphate buffer, pH 7.2 using the conditions mentioned above. b. Rinse in phosphate buffer. 2 × 60 min

310

➫ RNase free ➫ Low melting temperature ➫ See Appendix B6.2.1. ➫ Or anti-fluorescein conjugated to alkaline phosphatase or peroxidase ➫ See Appendix B2.15. ➫ See Appendix B2.5. ➫ See Appendix B2.8. ➫ See Appendix B2.7. ➫ dNTP ➫ See Appendix B2.12. ➫ See Appendix B2.19. ➫ See Appendix B4.3.2. ➫ See Appendix B3.4.3.

➫ According to the size of the sample ➫ Addition of a low percentage of glutaraldehyde (e.g., 0.1%) (see Section 2.1.3.3)

10.5 10.5.2.3

Indirect in Situ RT-PCR on Vegetable Tissue Using Floating Vibratome Sections

Sections

a. Embed in 5% agarose. b. Cool on ice. c. Adhere the sample to the object-holder of the vibratome. d. Immerse the sample in 2X SSC to obtain 80to 100-µm-thick floating sections. e. Remove the agarose mechanically by hand. f. Place two to five sections in Tris–HCl/NaCl/ CaCl2 buffer in microtubes.

10.5.2.4

➫ RNase free ➫ Use cyanolit-type glue. ➫ The area of the section must be large enough not to hamper subsequent procedures. ➫ All the different RT, PCR, and hybridization steps are carried out on floating sections in microtubes.

Pretreatments

a. Incubate the sections in Tris–HCl/NaCl/CaCl2 buffer. b. Add proteinase K.

5 min 37°°C 2 µg/ml 15 min 37°°C rt

c. Rinse in Tris–HCl/NaCl/CaCl2 buffer. d. Transfer the sections to other microtubes containing 0.1 M phosphate buffer, pH 7.4. 10.5.2.5

Reverse transcription

❶ Reaction mixture a. In a sterile microtube, prepare the reaction mixture: • 5X RT buffer 20 µ l • 0.1 M DTT 10 µ l • 10 mM dNTPs 5 µl • 40 U/µl RNasin 2.5 µ l • 10 µM anti-sense primer 10 µ l • Sterile water 47.5 µ l b. Replace the phosphate buffer with the reaction mixture, and pipette carefully to suspend the sections. c. Add 200 U/µl reverse 5 µl transcriptase. d. Incubate in a thermocycler. 1h 37°°C e. Deactivate the enzyme. 2 min 94°°C ❷ Washing The sections are rinsed in 5 min phosphate buffer.

➫ ➫ ➫ ➫ ➫ ➫

Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/µl Final concentration: 1 µM To a final volume of 95 µ l

➫ Final concentration: 10 U/µl ➫ Final volume: 100 µ l ➫ If MMLV is the reverse transcriptase used ➫ The temperature at which the enzyme is destroyed

311

Typical Protocols 10.5.2.6

Amplification

❶ Reaction mixture a. In a sterile microtube placed in ice, prepare the reaction mixture: • 10X PCR buffer 10 µ l • 25 mM MgCl2 6 µl • 10 mM dNTP mixture 5 µl • 10 µM sense primer 10 µ l • 10 µM anti-sense primer 10 µ l • Sterile water 55 µ l b. Replace the phosphate buffer with the reaction mixture, and suspend the sections by careful pipetting. ❷ The hot start

a. Incubate. b. Add 5 U/µl Taq DNA polymerase, and check that the tube is closed.

5 min 82°°C 4 µl

❸ Amplification cycles a. Program 10 amplification cycles: • Denaturing • Hybridization

• Extension b. Perform final extension. c. Stop the reaction.

Final concentration: 1X Final concentration: 1.5 mM Final concentration: 0.2 mM Final concentration: 1 µM Final concentration: 1 µM To a final volume of 96 µ l

➫ The specificity of primer matching must be ensured. There is a high risk of nonspecific hybridization at low temperature. ➫ Final concentration: 0.2 U/µl ➫ Final volume: 100 µ l ➫ To facilitate the subsequent washing steps, it is best not to add mineral oil. ➫ The number of cycles must be optimized for each operation.

1 min 94°°C 90 s 50°°C 90 s 72°°C 5 min 72°°C 10 s 30°°C

❹ Washing a. Remove all the reaction medium. b. Suspend the sections in 2 × 10 min phosphate buffer. ❺ Postfixation • 4% paraformaldehyde in 10 min 2X SSC buffer • Rinsing in 2X SSC 3 × 5 min

312

➫ ➫ ➫ ➫ ➫ ➫

➫ The hybridization temperature must be optimized in keeping with the characteristics of the primers used.

➫ The sections can be stored at 4°C in a thermocycler.

➫ To fix the hybrids that have been formed, and to stabilize the tissue and cell structures

10.5 10.5.2.7

Indirect in Situ RT-PCR on Vegetable Tissue Using Floating Vibratome Sections

Hybridization

The amplified products are detected by hybridization, using two cDNA probes labeled with digoxygenin. ❶ Reaction mixture a. In a sterile microtube placed in ice, prepare the reaction mixture: • 20X SSC 100 µ l • Deionized formamide 250 µ l • 50X Denhardt’s solution 10 µ l • 10 mg/ml tRNA 12.5 µ l • 10 mg/ml salmon sperm DNA 12.5 µ l • Labeled sense probe 8 µl • Labeled anti-sense probe

8 µl

• Sterile water 99 µ l b. Replace the phosphate buffer with the reaction mixture. c. Suspend the sections. d. Denature. 5 min 95°°C e. Cool immediately on ice. 5 min f. Incubate. Overnight 55–60°°C ❷ Washing • 2X SSC

• 1X SSC • TE/NaCl • RNase A treatment— 100 µg/ml in TE/NaCl • TE/NaCl • 1X SSC • 0.5X SSC • 0.1X SSC

30 min rt 30 min rt 30 min 37°°C 45–60 min 37°°C 3 × 10 min 30 min 60°°C 30 min rt 30 min rt

➫ See Chapter 6. ➫ Sense and anti-sense probes labeled by PCR ➫ Fluorescein or biotin.

➫ Final concentration: 4X ➫ Final concentration: 50% ➫ Final concentration: 1X ➫ Final concentration: 250 µg/ml ➫ Final concentration: 250 µg/ml ➫ Final concentration: 0.01 to 0.1 µg/ml of hybridization buffer ➫ Final concentration: 0.01 to 0.1 µg/ml of hybridization buffer ➫ To a final volume of 500 µ l

➫ On a heating block ➫ To stabilize the DNA in single-strand form ➫ In the thermocycler ➫ .The hybridization temperature must take into account the characteristics of the probes. ➫ See Chapter 6, Figure 6.4. ➫ It is always possible to increase or decrease the concentration of the SSC buffer and the washing time in line with the results obtained.

➫ The duration and temperature can be increased to reduce background. ➫ The temperature can be increased to reduce background

313

Typical Protocols 10.5.2.8

Immunocytological detection

❶ Direct detection by a conjugated antibody directed against the antigenic molecule used a. Incubate with: >2 h • Mouse anti-label monoclonal antibody conjugated to alkaline rt phosphatase at a dilution of 1:50 in Tris–HCl/NaCl buffer added to a blocking agent and a detergent b. Rinse: • In the same buffer, but 2 × 30 min without detergent ❷ Phosphatase alkaline detection a. Incubate: • Tris–HCl/NaCl/MgCl2 buffer, 5 min pH 9.6 b. Prepare the substrate extemporaneously:

• NBT • BCIP • Tris–HCl/NaCl/MgCl2 buffer, pH 9.6

30 µ l 40 µ l 10 ml

c. Incubate until a signal 10 min–2 h is obtained. rt d. Stop the reaction by washing 15 min in Tris–HCl buffer. ❸ Mounting the sections • In an aqueous medium if the substrate is NBT/BCIP or Fast Red • In a synthetic medium after dehydration in alcohol, and baths of solvent if the substrate is DAB 10.5.2.9

Observations

The sections are observed by light microscopy.

314

➫ According to the label ➫ On drops of reagent, with the section placed on the liquid ➫ Digoxygenin, fluorescein, or biotin ➫ Example: 0.5% ovalbumin ➫ Example: 0.05% Tween 20 ➫ Or more

➫ DAB in the case of detection with an antibody conjugated with peroxidase (see Chapter B6.2.2.2). ➫ Final concentration: 0.3 mg/ml ➫ Final concentration: 0.2 mg/ml ➫ To inhibit the phosphatases that are endogenous to certain types of tissue, necessary to add levamisol (1 mM) to this preparation ➫ In the same way, hydrogen peroxide (H2O2) used to inhibit endogenous peroxidases ➫ Detection carried out in darkness under a microscope

➫ Aquamount or Glycergel (see Appendix B8.1) ➫ Entellan, Eukitt (see Appendix B8.2)

10.6

Indirect in Situ RT-PCR Using Electron Microscopy

10.5.2.10 Controls ❶ Positive controls • Tissue containing the mRNA being sought ❷ Negative controls • Tissue that is known not to contain the mRNA being sought ❸ Reaction controls • Omission of reverse transcriptase, or treatment of tissue with RNase • Omission of Taq DNA polymerase • Omission of primers

• Omission of the antibody ❹ Diffusion control • 10 µl of the reaction medium is removed after amplification, and subjected to electrophoresis.

10.6

➫ This is the only way to find out if the reaction has taken place normally in the case of negative results. ➫ To confirm a specific amplification ➫ Control of the reverse transcription ➫ If the amplification step omitted, no positive result ➫ Possible that a weak signal may, however, appear, due to the endogenous reparative power of Taq DNA polymerase ➫ Detection control ➫ If the amplified products have diffused, a band will be clearly visible on the gel.

INDIRECT IN SITU RT-PCR USING ELECTRON MICROSCOPY ➫ See Chapter 11, Figures 11.12 through 11.17.

In situ RT-PCR using electron microscopy is currently one of the methods of choice for visualizing mRNA that is weakly expressed in a precise subcellular compartment. More than for any other approach, this identification necessitates high morphological quality, and the protocols must take account of this necessity, as in the example given here, where the use of thick, fixed tissue sections made on a vibratome ensures a high detection threshold in a wellconserved cell structure.

➫ Gloves must be worn. All the products must be RNase-free, the solutions must be prepared in DEPC water (see Appendix B1.2), and all equipment must be sterilized (see Appendix A2). ➫ In the case of RNA, which breaks down very easily, these conditions must be even more strictly respected.

315

Typical Protocols

10.6.1 Diagram of the Different Steps

10.6.2 Typical Protocol 10.6.2.1

Solutions

5% aqueous uranyl acetate 10 mg/ml salmon sperm DNA Blocking agent Anti-species antibody conjugated to colloidal gold Anti-digoxygenin conjugated to alkaline phosphatase or peroxidase 316

➫ See Appendix B7.2.1.2. ➫ See Appendix B2.8. ➫ See Appendix B6.2.1.

10.6

Indirect in Situ RT-PCR Using Electron Microscopy

Anti-fluorescein conjugated to alkaline phosphatase or peroxidase 0.4 mM biotin-14-dATP 2 mM CaCl2 50X Denhardt’s solution Digoxygenin-11-dATP 0.1 M DTT 10 mM dTTP, dCTP, dGTP, dATP Ethanol 100° (−20°C) Ethanol 70°, 95°, 100° Fluorescein-11-dATP 2.5% glutaraldehyde Igepal CA-630 LR-White 6 mM; 50 mM MgCl2 9‰ NaCl 4% paraformaldehyde PBS PBS-glycerol 0.1 M phosphate buffer 10X PCR buffer Proteinase K 10 µM anti-sense primer 10 µM sense primer Anti-sense probe Sense probe 10 mg/ml tRNA 40 U/µl RNasin 5X RT buffer 20X SSC Streptavidin conjugated to colloidal gold 5 U/µl Taq DNA polymerase Tris–HCl buffer Tris–HCl/CaCl2 buffer Tris–HCl/NaCl buffer Tris-HCl/NaCl/MgCl2 buffer DEPC water Sterile water 200 U/µl reverse transcriptase 10.6.2.2

➫ See Appendix B2.7. ➫ dNTP

➫ See Appendix B4.2. ➫ ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B5.3.2. See Appendix B2.12. See Appendix B2.19. See Appendix B4.3.2. See Appendix B3.4.3. See Appendix B8.1.1. See Appendix B3.4.1.

➫ ➫ ➫ ➫ ➫ ➫

See Appendix B2.14.1. See Section 5.3.2. See Section 5.3.2. See Section 6.3. See Section 6.3. See Appendix B2.15.

➫ See Appendix B3.5. ➫ ➫ ➫ ➫ ➫ ➫

See Appendix B3.7.1. See Appendix B3.7.2. See Appendix B3.7.5. See Appendix B3.7.4. See Appendix B1.2. See Appendix B1.1.

Fixation

a. Fix the sample in 4% 2h paraformaldehyde in phosphate buffer, using the conditions mentioned above. b. Rinse in phosphate buffer. 2 × 5 min 10.6.2.3

➫ See Appendix B2.3. ➫ See Appendix B2.5.

➫ According to the size of the sample ➫ Addition of a low percentage of glutaraldehyde (e.g., 0.1%) (see Section 2.1.3.3)

Sections

a. Adhere the sample to the object holder of a vibratome.

➫ Use cyanolit-type glue.

317

Typical Protocols b. Immerse the sample in 2X SSC buffer to obtain 50- to 70-µm-thick floating sections. c. Place two to five sections in 2X SSC buffer in microtubes.

10.6.2.4

➫ The area of the section must be large enough not to hamper subsequent procedures. ➫ All the different RT, PCR, and hybridization steps are carried out on floating sections in microtubes.

Pretreatments

a. Incubate the sections in Tris–HCl/NaCl/CaCl2 buffer. b. Add proteinase K.

5 min 37°°C 5 µg/ml 15 min 37°°C rt

c. Rinse in Tris-HCl/NaCl/CaCl2 buffer. d. Transfer the sections to other microtubes containing 0.1 M phosphate buffer, pH 7.4. 10.6.2.5

Reverse transcription

❶ Reaction mixture a. In a sterile microtube, prepare the reaction mixture: • 5X RT buffer 20 µ l • 0.1 M DTT 10 µ l • 10 mM dNTPs 5 µl • 40 U/µl RNasin 2.5 µ l • 10 µM anti-sense primer 10 µ l • Sterile water 47.5 µ l b. Replace the phosphate buffer with the reaction mixture, and pipette carefully to suspend the sections. c. Add 200 U/µl reverse 5 µl transcriptase. d. Incubate in a thermocycler. 1h 37°°C 1h 42°°C e. Deactivate the enzyme. 2 min 94°°C ❷ Washing The sections are rinsed in 5 min phosphate buffer. 10.6.2.6

Final concentration: 1X Final concentration: 10 mM Final concentration: 0.5 mM Final concentration: 1 U/µl Final concentration: 1 µM To a final volume of 95 µ l

➫ Final concentration: 10 U/µl ➫ Final volume: 100 µ l ➫ If MMLV is the reverse transcriptase used ➫ If AMV is the reverse transcriptase used ➫ The temperature at which the enzyme is destroyed

Amplification

❶ Reaction mixture a. In a sterile microtube placed in ice, prepare the reaction mixture: • 10X PCR buffer 10 µ l • 25 mM MgCl2 6 µl • 10 mM dNTP mixture 5 µl 318

➫ ➫ ➫ ➫ ➫ ➫

➫ Final concentration: 1X ➫ Final concentration: 1.5 mM ➫ Final concentration: 0.2 mM

10.6 • 10 µM labeled sense primer

Indirect in Situ RT-PCR Using Electron Microscopy

10 µ l

• 10 µM labeled anti-sense primer 10 µ l • Sterile water 55 µ l b. Replace the phosphate buffer with the reaction mixture, and suspend the sections by careful pipetting. ❷ The hot start

a. Incubate b. Add 5 U/µl Taq DNA polymerase, and check that the tube is closed.

5 min 82°°C 4 µl

❸ Amplification cycles a. Program 20 amplification cycles: • Denaturing • Hybridization

• Extension b. Perform final extension. c. Stop the reaction.

➫ Final concentration: 1 µM ➫ Possible to use only one labeled primer, but to this extent the effectiveness of the amplification will decline ➫ Final concentration: 1 µM ➫ To a final volume of 96 µ l

➫ The specificity of primer matching must be ensured. There is a high risk of nonspecific hybridization at low temperature. ➫ Final concentration: 0.2 U/µl ➫ Final volume: 100 µ l ➫ To facilitate the subsequent washing steps, it is best not to add mineral oil. ➫ The number of cycles must be optimized for each operation.

1 min 94°°C 90 s 50°°C 90 s 72°°C 5 min 72°°C 10 s 30°°C

➫ The hybridization temperature must be optimized in keeping with the characteristics of the primers being used.

➫ The sections can be stored at 4°C in a thermocycler.

❹ Washing a. Remove all the reaction medium. b. Suspend the sections in 2 × 10 min phosphate buffer. 10.6.2.7

Hybridization

The amplified products are detected by hybridization, using two specific oligonucleotidic probes labeled with digoxygenin. ❶ Reaction mixture a. In a sterile microtube placed in ice, prepare the reaction mixture: • 20X SSC 100 µ l • Deionized formamide 250 µ l • 50X Denhardt’s solution 10 µ l • 10 mg/ml tRNA 12.5 µ l

➫ See Chapter 6. ➫ Fluorescein or biotin

➫ ➫ ➫ ➫

Final concentration: 4X Final concentration: 50% Final concentration: 1X Final concentration: 250 µg/ml

319

Typical Protocols • 10 mg/ml salmon sperm DNA 12.5 µ l • Labeled sense probe 8 µl (1.25 pmol/µl) • Labeled anti-sense probe 8 µl (1.25 pmol/µl) • Sterile water 99 µ l b. Replace the phosphate buffer with the reaction mixture. c. Suspend the sections. d. Denature. 5 min 95°°C e. Cool immediately on ice. 5 min f. Incubate. Overnight 40°°C

❷ Washing • 2X SSC • 1X SSC • 0.5X SSC ❸ Postfixation • 4% paraformaldehyde in 2X SSC buffer • Rinsing in 2X SSC 10.6.2.8

➫ Final concentration: 20 pmol/ml ➫ To a final volume of 500 µ l

➫ On a heating block ➫ To stabilize the DNA in single-strand form ➫ In the thermocycler ➫ Necessary that the hybridization temperature take into account the characteristics of the probes ➫ See Chapter 6, Figure 6.4.

30 min rt 30 min rt 30 min rt

➫ It is always possible to increase or decrease the concentration of the SSC buffer and the washing time in line with the results obtained.

10 min

➫ To fix the hybrids that have been formed, and to stabilize the tissue and cell structures

3 × 5 min

Embedding

Vibratome sections are embedded in LR-White resin, or any other hydrophilic resin. ❶ Dehydration • Alcohol 50°, 70°, 95°, 100° 10 min per bath ❷ Substitution • Alcohol 100°–LR White 30 min (2:1 v/v) • Alcohol 100°–LR White 30 min (1:1 v/v) • Alcohol 100°–LR White 30 min (1:2 v/v) ❸ Polymerization The sections, spread on a flat 2 days surface in a drop of resin, 60°°C are covered with a capsule.

320

➫ Final concentration: 250 µg/ml ➫ Final concentration: 20 pmol/ml

➫ Of the Unicryl or Lowicryl type

➫ Polymerization can take place only in anaerobic conditions. ➫ See Section 8.11.

10.6 10.6.2.9

Indirect in Situ RT-PCR Using Electron Microscopy

Ultramicrotomy

In this procedure, 100-nm sections are made on an ultracut equipped with a diamond knife, and placed on collodionized, carbonated nickel grids. 10.6.2.10

Immunocytological detection

❶ Indirect detection by an antibody directed against the antigenic molecule used a. Incubate: • Mouse anti-label monoclonal 1h antibody at a dilution of 1:50 in rt Tris–HCl/NaCl buffer added to a blocking agent and a detergent b. Rinse: • In the same buffer, but without detergent • In Tris–HCl/NaCl buffer, pH 8.2, added to a blocking agent and a detergent

2 × 5 min

➫ A pH of 8.2 is important for the stability of an antibody conjugated to colloidal gold. ➫ Example: 0.5% ovalbumin ➫ Example: 0.05% Tween 20 ➫ Depending on the primary antibody used ➫ The intensity of the labeling inversely proportional to the size of the gold particles

. ➫ Nonsterile, but filtered on 0.2 µm ➫ Nonsterile, but filtered on 0.2 µm

Observations

The grids are observed by transmission electron microscopy. 10.6.2.12

➫ According to the label ➫ The grids are incubated on drops of reagent, with the section placed on the liquid. ➫ Digoxygenin, fluorescein, or biotin ➫ Example: 0.5% ovalbumin ➫ Example: 0.05% Tween 20

2 × 5 min

c. Incubate: • Anti-mouse antibody conjugated 1h to 10 nm colloidal gold at a rt dilution of 1:50 in the same buffer d. Rinse: • In Tris–HCl/NaCl buffer, 2 × 5 min pH 8.2 • In 2X SSC buffer 2 × 5 min ❷ Fixation • 2.5% glutaraldehyde in 2X SSC 5 min buffer • Washing in 2X SSC 2 × 5 min • Rapid rinsing in distilled water ❸ Contrast • By 5% aqueous uranyl acetate 30 min • Rinsing in distilled water • Drying the grids 10.6.2.11

➫ See Section 8.11.4.

➫ The observation voltage must be low, i.e., 60 to 80 kV.

Controls

❶ Positive controls • Tissue containing the mRNA being sought

➫ The only way to find out if the reaction has taken place normally in the case of negative results 321

Typical Protocols ❷ Negative controls • Tissue that is known not to contain the mRNA being sought ❸ Reaction controls • Omission of reverse transcriptase, or treatment of tissue with RNase • Omission of Taq DNA polymerase • Omission of primers

• Omission of the primary antibody ❹ Diffusion control • 10 µl of the reaction medium is removed after amplification, and subjected to electrophoresis.

322

➫ To confirm a specific amplification ➫ Control of the reverse transcription ➫ If the amplification step omitted, no positive result ➫ Possible that a weak signal may, however, appear, due to the endogenous reparative power of Taq DNA polymerase ➫ Detection control ➫ If the amplified products have diffused, a band will be clearly visible on the gel.

Chapter 11 Examples of Observations

❽ Detection • In situ hybridization and/or • Immunohistochemistry ➒ Bar ❿ Comments

• C: Indirect in situ PCR

❸ Tissue ❹ Fixation ➎ Tissue preparation • Paraffin embedding • Sections ❻ Pretreatment • Proteinase K ❼ Amplification • A: Control by in situ hybridization • B: Direct in situ PCR

❷ Methods

❶ Title

FIGURE 11.1 (Color Figure 11.1 follows page 336.)

Fluorescent cDNA probe 20-mer sense and anti-sense primers Incorporation of dUTP-11-biotin 25 cycles 20-mer sense and anti-sense primers 25 cycles ➫ Alkaline phosphatase conjugated anti-fluorescein ➫ Streptavidin/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ The amplified product (dark purple precipitate) is detected on three adjacent sections in the cells of the cervical epithelium, which have perinuclear halos and atypical nuclei. Furthermore, no viral DNA is visible in the basal cells after in situ hybridization alone (A). All the cells are strongly positive after direct in situ PCR (B). The signal is more specific after indirect in situ PCR (C).

➫ ➫ ➫ ➫ ➫ ➫

➫ 5 µg/ml

➫ Adjacent 4-µm sections

➫ A comparison between indirect in situ hybridization and direct and indirect in situ PCR for the detection of human papilloma virus (HPV) DNA ➫ A: In situ hybridization ➫ B: Direct in situ PCR ➫ C: Indirect in situ RT-PCR ➫ Squamous intraepithelial lesion (human condyloma) ➫ 4% paraformaldehyde

Examples of Observation

325

326 • B: Indirect in situ PCR • C: Control without Taq ADN polymerase ❼ Detection • Immunohistochemistry ❽ Bar ➒ Comments

Title Method Monolayer culture Fixation Pretreatment • Proteinase K ❻ Amplification • A: Direct in situ PCR

❶ ❷ ❸ ❹ ➎

FIGURE 11.2 (Color Figure 11.2 follows page 336.) Detection of HPV 6 DNA in HeLa cells culture A: Direct in situ PCR HeLa cells 4% paraformaldehyde

Biotinylated dATP 20-mer sense and anti-sense primers Incorporation of dUTP-11-biotin 25 cycles 20-mer sense and anti-sense primers 25 cycles

➫ Streptavidin/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ HeLa cells contain, on average, more than ten copies of the virus per genome. The direct (A) and the indirect (B) method give a strong signal in all the cells. No signal is found when Taq polymerase is omitted (C).

➫ ➫ ➫ ➫ ➫ ➫

➫ 1 µg/ml

➫ ➫ ➫ ➫

Examples of Observations

❼ Detection • In situ hybridization, and • Immunohistochemistry ❽ Bar ➒ Comments

Method Monolayer culture Fixation Pretreatment • Proteinase K ❻ Amplification • Indirect in situ PCR

❷ ❸ ❹ ➎

❶ Title

FIGURE 11.3 (Color Figure 11.3 follows page 336.)

➫ Biotinylated HPV 6 cDNA probe ➫ Streptavidin/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ Only the HeLa cells, which are characterized by their fibroblastic shapes, have positive nuclei (see Figure 11.2). Those of the Hep2 cells, which are characterized by their epidermoid appearance, remain negative after amplification. This internal control demonstrates the specificity of the detection method.

➫ 20-mer sense and anti-sense primers ➫ 25 cycles

➫ 1 µg/ml

➫ The detection of HPV 6 DNA in a coculture of two different cell types ➫ Indirect in situ PCR ➫ HeLa and Hep2 cells ➫ 4% paraformaldehyde

Examples of Observation

327

328 • D: Control ❽ Detection • In situ hybridization, and • Immunohistochemistry ➒ Bar ❿ Comments

❷ Method ❸ Tissue ❹ Tissue preparation • Frozen sections ➎ Fixation ❻ Pretreatment • Proteinase K ❼ Amplification • A: Indirect in situ RT-PCR • B: Reverse transcription • C: In situ hybridization

❶ Title

FIGURE 11.4 (Color Figure 11.4 follows page 336.)

30-mer anti-sense probe 25-mer anti-sense probe 25-mer sense and anti-sense probes 25 cycles Treatment by RNase ➫ 30-mer sense and anti-sense oligonucleotide probes ➫ Antibody/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ After the indirect RT-PCR reaction (A), the signal is very strong and it is important to note that there are still some negative cells. A little more than half the cells in the anterior lobe of the pituitary are detected after the RT reaction (B) and after in situ hybridization (C). After the destruction of the RNA before the RTPCR, no signal is detectable (D).

➫ ➫ ➫ ➫ ➫

➫ 1 µg/ml

➫ 7 µm ➫ 4% paraformaldehyde

➫ A demonstration of the specificity of the detection of the mRNA that codes for the growth hormone receptor in the pituitary gland ➫ Indirect in situ RT-PCR ➫ Rat pituitary

Examples of Observations

• Immunohistochemistry ➒ Bar ❿ Comments

❷ Method ❸ Tissue ❹ Tissue preparation • Paraffin-embedded sections ➎ Fixation ❻ Pretreatment • Proteinase K ❼ Amplification • A: 5 cycles • B: 15 cycles • C: 25 cycles ❽ Detection • In situ hybridization, and

❶ Title

FIGURE 11.5 (Color Figure 11.5 follows page 336.)

➫ 30-mer sense and anti-sense probes labeled with digoxigenin ➫ Anti-digoxigenin/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ The number of cycles has no effect on the percentage of positive cells observed (see Figure 11.4), but a higher number of cycles produces a stronger signal: A (5 cycles) < B (15 cycles) < C (25 cycles).

➫ 3 µg/ml ➫ 25-mer sense and anti-sense primers

➫ 5 µm ➫ 4% paraformaldehyde

➫ The effect of the number of reaction cycles on the amplification of the mRNA that codes for the growth hormone receptor in the pituitary ➫ Indirect in situ RT-PCR ➫ Rat pituitary

Examples of Observation

329

330 • Immunohistochemistry ➒ Bar ❿ Comments

❷ Method ❸ Tissue ❹ Tissue preparation • Paraffin-embedded sections ➎ Fixation ❻ Pretreatment • Proteinase K ❼ Amplification • A: Indirect in situ RT-PCR • B: Control without amplification ❽ Detection • In situ hybridization

❶ Title

FIGURE 11.6 (Color Figure 11.6 follows page 336.)

➫ 30-mer sense and anti-sense probes labeled with digoxigenin ➫ Anti-digoxigenin/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ GH mRNA synthesis occurs in the lymphoid white pulp that surrounds and follows the arteries, and in the lymphocytes of the red pulp. The reticular cells of the connective network remain negative (A). No signal is detected in the control (B). (From Recher, S. et al., J. Histochem. Cytochem., 49, 347, 2001. With permission.)

➫ 25-mer sense and anti-sense primers ➫ Omission of the RT step and Taq DNA polymerase

➫ 3 µg/ml

➫ 5 µm ➫ 4% paraformaldehyde

➫ The detection of weakly expressed mRNA coding for extrapituitary growth hormone (GH) in a lymphoid organ, namely, the spleen ➫ Indirect in situ RT-PCR ➫ Rat spleen

Examples of Observations

• Immunohistochemistry ➒ Bar ❿ Comments

❽ Detection • In situ hybridization, and

❹ Tissue preparation • Paraffin-embedded sections ➎ Fixation ❻ Pretreatment • Proteinase K ❼ Amplification • Indirect in situ RT-PCR

❷ Method ❸ Tissue

❶ Title

FIGURE 11.7 (Color Figure 11.7 follows page 336.)

➫ 30-mer sense and anti-sense probes labeled with digoxigenin ➫ Anti-digoxigenin/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ GH-gene-expressing cells are mainly found in the thymic cortex, while the reticular and epithelial cells of the medulla are negative, and act as an internal control (A). In Peyer’s patches, the signal is specifically associated with all the lymphocytes, whereas epithelial cells of the terminal ileum remained negative (B). (From Recher, S. et al., J. Histochem. Cytochem., 49, 347, 2001. With permission.)

➫ 25-mer sense and anti-sense primers ➫ 25 cycles

➫ 3 µg/ml

➫ 5 µm ➫ 4% paraformaldehyde

➫ GH gene expression in adult rat thymus and Peyer’s patches ➫ Indirect in situ RT-PCR ➫ A: Adult rat thymus ➫ B: A Peyer’s patch in an adult rat

Examples of Observation

331

332 • Immunohistochemistry ➒ Bar ❿ Comments

❽ Detection • In situ hybridization, and

❷ Method ❸ Tissue ❹ Tissue preparation • Paraffin-embedded sections ➎ Fixation ❻ Pretreatment • Proteinase K ❼ Amplification • A: Indirect in situ RT-PCR • B: Control without amplification

❶ Title

FIGURE 11.8 (Color Figure 11.8 follows page 336.)

➫ 30-mer sense and anti-sense olidonucleotide probes labeled with digoxigenin ➫ Anti-digoxigenin/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ GH gene expression is found in all the fetal thymus cells. However, the lymphocytes located in the cortical region of the thymus lobe give a stronger signal (A). In situ RT-PCR performed without Taq DNA polymerase on an adjacent section is used as a negative control (B). (From Recher, S. et al., J. Histochem. Cytochem., 49, 347, 2001. With permission.)

➫ 25-mer anti-sense primer ➫ 25-mer sense and anti-sense primers ➫ Omission of Taq DNA polymerase

➫ 3 µg/ml

➫ 5 µm ➫ 4% paraformaldehyde

➫ Detection of mRNA coding for the growth hormone in the rat fetal thymus ➫ Indirect in situ RT-PCR ➫ 18-day-old rat fetal thymus

Examples of Observations

• Immunohistochemistry ➒ Bar ❿ Comments

❷ Method ❸ Tissue ❹ Tissue preparation • Paraffin-embedded sections ➎ Fixation ❻ Pretreatment • Proteinase K ❼ Amplification • RT • Indirect in situ PCR ❽ Detection • In situ hybridization, and

❶ Title

FIGURE 11.9 (Color Figure 11.9 follows page 336.)

➫ 30-mer sense and anti-sense oligonucleotide probes labeled with digoxigenin ➫ Anti-digoxigenin/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ In the fetus, the liver has a hematopoietic function. Here, a strong signal demonstrating GH gene expression is detected in the hematopoietic cells around the centrolobular vein, whereas the hepatocytes remain consistently negative. (From Recher, S. et al., J. Histochem. Cytochem., 49, 347, 2001. With permission.)

➫ 25-mer anti-sense primer ➫ 25-mer sense and anti-sense primers

➫ 3 µg/ml

➫ 5 µm ➫ 4% paraformaldehyde

➫ The detection of mRNA coding for the growth hormone (GH) in rat fetal liver ➫ Indirect in situ RT-PCR ➫ 18-day-old rat fetal liver

Examples of Observation

333

334 • Immunohistochemistry ➒ Bar ❿ Comments

❷ Method ❸ Tissue ❹ Tissue preparation • Paraffin-embedded sections ➎ Fixation ❻ Pretreatment • Proteinase K ❼ Amplification • A: RT Indirect in situ PCR • B: Omission of Taq DNA polymerase ❽ Detection • In situ hybridization, and

❶ Title

FIGURE 11.10 (Color Figure 11.10 follows page 336.)

➫ 30-mer sense and anti-sense oligonucleotide probes labeled with digoxigenin ➫ Anti-digoxigenin/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ All the proliferative epithelial cells and fibroblasts are positive (A). The specificity of the reaction is demonstrated by the absence of a signal when Taq DNA polymerase is omitted (B).

➫ 25-mer anti-sense primer ➫ 25-mer sense and anti-sense primers ➫ Control without amplification

➫ 3 µg/ml

➫ 5 µm ➫ 4% formol

➫ The location of mRNA coding for extrapituitary growth hormone in a benign mammary pathology, namely, fibroadenoma ➫ Indirect in situ RT-PCR ➫ Fibroadenoma in a human mammary gland

Examples of Observations

• Immunohistochemistry ➒ Bar ❿ Comments

❷ Method ❸ Tissue • A: Secondary root • B: Degenerative nodule ❹ Tissue preparation • Vibratome sections ➎ Fixation ❻ Pretreatment • Proteinase K ❼ Amplification RT Indirect in situ PCR ❽ Detection • In situ hybridization, and

❶ Title

FIGURE 11.11 (Color Figure 11.11 follows page 336.)

➫ cDNA sense and anti-sense probes labeled with digoxigenin ➫ Anti-digoxigenin/alkaline phosphatase/NBT-BCIP ➫ 10 µm ➫ Only the mesenchymatous cells in the secondary root give a positive signal (A), whereas the cells in a degenerative nodule are negative (B). (From De Billy, F., unpublished data. With permission.)

➫ 25-mer anti-sense primer ➫ 25-mer sense and anti-sense primers

➫ 2 µg/ml

➫ 70 µm ➫ 4% paraformaldehyde

➫ The detection of mRNA coding for leghemoglobin in a secondary root ➫ Indirect in situ RT-PCR ➫ Roots of Medicago truncatula

Examples of Observation

335

FIGURE 11.12

336 • Immunocytochemistry ➒ Bar ❿ Comments

• B: Omission of Taq DNA polymerase ❽ Detection • In situ hybridization, and

❸ Tissue ❹ Fixation ➎ Tissue preparation • Vibratome sections ❻ Pretreatments • Proteinase K • Triton X100 ❼ Amplification • A: RT Indirect in situ PCR

❷ Method

❶ Title

25-mer anti-sense primer 25-mer sense and anti-sense primers 20 cycles Control without amplification ➫ Before embedding ➫ 30-mer sense and anti-sense probes labeled with digoxigenin ➫ Antibody/peroxidase/DAB ➫ 10 µm ➫ Only a few cells are positive in this semithin transversal section of the thick section of the anterior lobe of the pituitary (insert). No signal (dense product) is visible in this section other than in the cytoplasm of certain cells (A). In the absence of amplification, there are no positive cells (B).

➫ ➫ ➫ ➫

➫ 1 µg/ml ➫ 0.01%

➫ 50 µm

➫ Detection, on semithin sections, of pituitary mRNA coding for the growth hormone receptor ➫ Indirect in situ RT-PCR in electron microscopy: Pre-embedding method ➫ Frontal lobe of rat pituitary ➫ 4% paraformaldehyde

Examples of Observations

FIGURE 11.13

❽ Detection • Immunocytology ➒ Bar ❿ Comments

In situ hybridization

• B: RT In situ PCR

❸ Tissue ❹ Fixation ➎ Tissue preparation • Hydrophilic resin embedding ❻ Pretreatment • Proteinase K ❼ Amplification • A: In situ hybridization

❷ Method

❶ Title

➫ Antibody/colloidal gold (10 nm) ➫ 1 µm ➫ The colloidal gold particles (arrow) are present in the cytoplasmic matrix, near the endoplasmic reticulum (er) of three cell types: gonadotrophs (LH-FSH), lactotrophs (PRL), and somatotrophs (GH). A comparison between the labeling obtained without amplification, after in situ hybridization (A) and after amplification (B), shows no significant increase in the density of the colloidal gold particles.

➫ 30-mer sense and anti-sense probes labeled with digoxigenin ➫ 25-mer anti-sense primer ➫ 25-mer sense and anti-sense primers ➫ 20 cycles ➫ 30-mer sense and anti-sense probes labeled with digoxigenin

➫ 1 µg/ml

➫ LR White

➫ Ultrastructural detection of mRNA coding for the growth hormone receptor in the pituitary ➫ Indirect in situ RT-PCR in electron microscopy. Hydrophilic resin postembedding method ➫ Rat pituitary ➫ 4% paraformaldehyde

Examples of Observation

337

FIGURE 11.14

338 ❽ Detection • Immunocytology ➒ Bar ❿ Comments

In situ hybridization

• B: RT In situ PCR

❸ Tissue ❹ Fixation ➎ Tissue preparation • Freezing • Cryoultramicrotomy ❻ Pretreatment ❼ Amplification • A: In situ hybridization

❷ Method

❶ Title

➫ Antibody/colloidal gold (10 nm) ➫ 1 µm ➫ The colloidal gold particles (arrows) are present in the same cells, close to the endoplasmic reticulum (er), both without amplification, after in situ hybridization alone (A), and after amplification (B). No significant increase in the density of colloidal gold particles is observed with this non-embedding amplification method.

➫ 30-mer sense and anti-sense probes labeled with digoxigenin ➫ 25-mer anti-sense primer ➫ 25-mer sense and anti-sense primers ➫ Limited to five cycles ➫ 30-mer sense and anti-sense probes labeled with digoxigenin

➫ Ultrathin frozen tissue sections (≈80 nm) ➫ None

➫ Ultrastructural detection of mRNA coding for the growth hormone receptor in the pituitary ➫ Indirect in situ RT-PCR in electron microscopy: Non-embedding method ➫ Rat pituitary ➫ 4% paraformaldehyde

Examples of Observations

FIGURE 11.15

➒ Bar ❿ Comments

• Immunocytology

❽ Detection • In situ hybridization

❸ Tissue ❹ Fixation ➎ Tissue preparation • Vibratome sections ❻ Pretreatments • Proteinase K • Triton X100 ❼ Amplification • RT • In situ PCR

❷ Method

❶ Title

➫ 30-mer sense and anti-sense probes labeled with digoxigenin ➫ After embedding in hydrophilic resin (LR White) ➫ Antibody/colloidal gold (10 nm) ➫ 1 µm ➫ The colloidal gold particles are present in the cytoplasmic matrix, close to the endoplasmic reticulum (er), in the gonadotrophs (LH-FSH), lactotrophs (PRL), and somatotrophs (GH). The density of the labeling is higher with this preembedding amplification method.

➫ 25-mer anti-sense primer ➫ 25-mer sense and anti-sense primers ➫ 20 cycles

➫ 1 µg/ml ➫ 0.01%

➫ 50 µm

➫ Ultrastructural detection of mRNA coding for the growth hormone receptor in the pituitary ➫ Indirect in situ RT-PCR in electron microscopy: Preembedding method ➫ Rat pituitary ➫ 4% paraformaldehyde

Examples of Observation

339

FIGURE 11.16

340 ➒ Bar ❿ Comments

• Immunocytology

❽ Detection • In situ hybridization

❸ Tissue ❹ Fixation ➎ Tissue preparation • Vibratome sections ❻ Pretreatments • Proteinase K • Triton X100 ❼ Amplification • RT • In situ PCR

❷ Method

❶ Title

➫ 30-mer sense and anti-sense probes labeled with digoxigenin ➫ After embedding in hydrophilic resin ➫ Antibody/colloidal gold (10 nm) ➫ 1 µm ➫ Colloidal gold particles are present in the cytoplasm of a somatotroph (GH), but not in any corticotrophs (ACTH) or thyrotrophs (insert), which means that the amplified products have not diffused between different types of cell.

➫ 25-mer anti-sense primer ➫ 25-mer sense and anti-sense primers ➫ 20 cycles

➫ 1 µg/ml ➫ 0.01%

➫ 50 µm

➫ Cellular specificity of the ultrastructural detection of the mRNA coding for the growth hormone receptor in the pituitary ➫ Indirect in situ RT-PCR in electron microscopy: Preembedding method ➫ Rat pituitary ➫ 4% paraformaldehyde

Examples of Observations

FIGURE 11.17

➒ Bar ❿ Comments

• Immunocytology

❸ Tissue ❹ Fixation ➎ Tissue preparation • Vibratome sections ❻ Pretreatments • Proteinase K • Triton X100 ❼ Amplification • RT • In situ PCR ❽ Detection • RT-PCR — In situ hybridization, and — Immunocytology

❷ Method

❶ Title

➫ 30-mer sense and anti-sense probes labeled with digoxigenin ➫ After embedding ➫ Antibody/colloidal gold (10 nm) ➫ Anti-prolactin (PRL) serum ➫ Colloidal gold (5 nm) ➫ 1 µm ➫ 10-nm colloidal gold particles that are used to visualize mRNA coding for the growth hormone are present in the cytoplasm and the nucleus (N) of a lactotroph (PRL), which is identified by the immunocytological detection of the content of its secretion grains (5-nm gold particles).

➫ 25-mer anti-sense primer ➫ 25-mer sense and anti-sense primers

➫ 1 µg/ml ➫ 0.01%

➫ 50 µm

➫ The double ultrastructural detection of mRNA coding for the growth hormone and prolactin in lactotrophs ➫ Indirect in situ RT-PCR in electron microscopy: Preembedding method ➫ Immunocytology after embedding ➫ Rat pituitary ➫ 4% paraformaldehyde

Examples of Observation

341

Appendices

CONTENTS

————————— A – EQUIPMENT ————————— A1 Practical Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A1.1 RNase-Free Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A1.2 Chemical Risks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A1.3 Radioactive Risks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A2 Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A3 Pretreatment of Slides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A4 Electrophoresis of PCR Products in Agarose Gel. . . . . . . . . . . . . . . .

349 349 350 351 351 352 353

————————— B – REAGENTS ————————— B1 Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B1.1 Sterile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B1.2 Diethylpyrocarbonate (DEPC) . . . . . . . . . . . . . . . . . . . . . . . . B2 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.1 Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.2 Ammonium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.3 Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.3.1 Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . B2.3.2 Calcium Chloride/Cobalt Chloride . . . . . . . . . . . . . B2.4 Deionized Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.5 Denhardt’s Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.6 Dextran Sulfate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.7 Dithiothreitol (DTT). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.8 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.9 DNase I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.10 Ethylene Diamine Tetra-Acetic Acid (EDTA) . . . . . . . . . . . . B2.11 Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.12 Magnesium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.13 Poly (A) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.14 Proteinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.14.1 Proteinase K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.14.2 Pepsine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.14.3 Pronase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.15 RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.16 RNase A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.17 Sarcosyl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.18 Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

355 355 355 355 355 356 356 356 357 357 357 358 358 359 359 359 360 360 360 360 360 361 361 361 362 362 362 345

Appendices

B3

B4

B5

B6

346

B2.19 Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.20 Sodium Hydroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.21 Tris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B2.22 Triton X-100. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.1 Acetylation Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.2 Cacodylate Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.3 DNase I Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.4 Phosphate Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.4.1 1 M Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.4.2 Phosphate/NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.4.3 PBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.5 SSC Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.6 TE (Tris–EDTA) Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.6.1 TE Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.6.2 TE–NaCl Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.7 Tris–HCl Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.7.1 Tris–HCl Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . B3.7.2 Tris–HCl/CaCl2 Buffer . . . . . . . . . . . . . . . . . . . . . . B3.7.3 Tris–HCl/Glycine Buffer. . . . . . . . . . . . . . . . . . . . . B3.7.4 Tris–HCl/MgCl2 Buffer . . . . . . . . . . . . . . . . . . . . . . B3.7.5 Tris–HCl/NaCl Buffer . . . . . . . . . . . . . . . . . . . . . . . B3.7.6 Tris–HCl/NaCl/MgCl2 Buffer . . . . . . . . . . . . . . . . . Fixatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.1 Formol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.1.1 Neutral Buffered Formalin . . . . . . . . . . . . . . . . . . . B4.1.2 Formol Saline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.2 Glutaraldehyde (2.5%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.3 Paraformaldehyde. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B4.3.1 Paraformaldehyde 40% . . . . . . . . . . . . . . . . . . . . . . B4.3.2 Paraformaldehyde 4% . . . . . . . . . . . . . . . . . . . . . . . B4.3.3 Paraformaldehyde 4%/Glutaraldehyde 0.05% . . . . Embedding Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.1 Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.2 Epoxy Resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.2.1 Epon–araldite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.2.2 Epon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.3 Acrylic Resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.3.1 Lowicryl K4M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . B5.3.2 LR White Medium. . . . . . . . . . . . . . . . . . . . . . . . . . B5.3.3 Unicryl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Revelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.1 Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.1.1 Standard Developer . . . . . . . . . . . . . . . . . . . . . . . . . B6.1.2 Fixative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.2 Immunocytology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.2.1 Blocking Solutions . . . . . . . . . . . . . . . . . . . . . . . . . B6.2.1.1 Nonspecific Sites . . . . . . . . . . . . . . . . . . B6.2.1.2 Endogenous Alkaline Phosphatases . . . B6.2.1.3 Endogenous Peroxidases . . . . . . . . . . . .

363 363 363 364 364 364 364 365 365 365 366 366 367 367 367 368 368 368 369 369 369 370 370 371 371 371 371 371 372 372 372 373 373 373 374 374 374 375 375 376 376 377 377 377 377 378 378 378 378 379

Appendices B6.2.2

Chromogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B6.2.2.1 Alkaline Phosphatase . . . . . . . . . . . . . . B6.2.2.2 Peroxidase . . . . . . . . . . . . . . . . . . . . . . . B7 Stains/Coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.1 Light Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.1.1 Cresyl Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.1.2 Eosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.1.3 Harris’s Hematoxylin . . . . . . . . . . . . . . . . . . . . . . . B7.1.4 Methylene Green . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.1.5 Rapid Nuclear Red . . . . . . . . . . . . . . . . . . . . . . . . . B7.1.6 Toluidine Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.2 Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.2.1 Uranyl Acetate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.2.1.1 2.5% Alcoholic Uranyl Acetate . . . . . . . B7.2.1.2 2 to 5% Aqueous Uranyl Acetate . . . . . B7.2.1.3 4% Neutral Uranyl Acetate . . . . . . . . . . B7.2.2 Lead Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.2.3 Methylcellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . B7.2.3.1 2% Methylcellulose. . . . . . . . . . . . . . . . B7.2.3.2 0.8% Methylcellulose, 0.2% Neutral Uranyl Acetate . . . . . . . . . . . . . B7.2.4 Sodium Silicotungstate (0.5%) . . . . . . . . . . . . . . . . B8 Mounting Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B8.1 Aqueous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B8.1.1 Buffered Glycerine . . . . . . . . . . . . . . . . . . . . . . . . . B8.1.2 Moviol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B8.2 Permanent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

379 379 380 380 380 380 381 381 382 382 382 383 383 383 383 383 384 384 384 385 385 386 386 386 386 387

347

Appendix A EQUIPMENT This appendix presents the procedures for preparing materials for in situ PCR/RT-PCR to obtain the best preservation of nucleic acids.

A1 PRACTICAL PRECAUTIONS A1.1

RNase-Free Conditions

 Working areas • Areas close to sterility  Probe • Use of all the probes

• Storage — DEPC-treated water — Choice of TE buffer (TE/NaCl)  Tissue • Sampling in sterile conditions • Storage of sections

— Sections of tissue embedded in paraffin

— Frozen sections

— Semithin frozen sections

— Ultrathin sections

➫ Reagents are available that destroy RNases. ➫ RNase activity is inhibited in the buffer ® (presence of RNasin ). ➫ Commercial solutions for inhibiting RNases are available. ➫ This buffer, which is very stable, inhibits the action of enzymes that break down DNA. ➫ Instruments must be sterile. ➫ Slides can be dried at room temperature. ➫ Storage in the absence of water is the best way of inhibiting RNases. RNases and DNases act only in the presence of water. ➫ Place the dry slides in a airtight box containing desiccant (silicagel). ➫ Prefer the storage in paraffin block. ➫ Place the dry slides in an airtight box containing a desiccant (silicagel) at room temperature or −20°C. Do not open the box until it is at room temperature, to limit condensation on the sections. ➫ At 4°C under a drop of sucrose used to pick up the sections ➫ Limited storage: 1 to 2 weeks ➫ Do not store these sections for more than 1 week.

349

Equipment  Equipment/Reagents/Solutions • Eppendorf tubes • Sterilized cones • Gloves • New reagents • Recipients

• Water treated with DEPC or containing RNase inhibitors  Conclusion Avoiding RNase contamination is easier than removing it.

➫ Disposable is preferable to sterilized equipment. ➫ Do not touch the equipment or the reagents without gloves. ➫ Reserved exclusively for in situ PCR/RTPCR. ➫ Reserved for in situ PCR/RT-PCR and sterilized immediately after use (otherwise, disposable equipment). ➫ The risk of contamination increases with time, in relation to the frequency of opening.

A1.2 Chemical Risks ❶ Reagents/Resins  Reagents For electron microscopy a number of products are used that are dangerous if touched or inhaled:

➫ In accordance with the manufacturer’s instructions ➫ All experiments must be carried out under a fume hood.

• Formaldehyde • Glutaraldehyde Some organic solvents must be used in a ventilated area that vents to the outside. Other products, such as: • • • •

Formamide Lead nitrate Sodium cacodylate Uranyl acetate

are highly toxic if inhaled or ingested. They must be used with great care.  Resins Epoxy and acrylic resins are carcinogenic and highly toxic. Skin contact is dangerous.

❷ Storage/Waste products Glassware, waste chemicals, and biological material for incineration are collected in special waste containers.

350

➫ Toxic vapors (isoamyl acetate, xylene, acetone, etc.)

➫ α radiation ➫ Any trace of these products on the skin must be carefully washed off in running water. ➫ Wear a mask. ➫ Avoid all contact with the skin. In the case of spills, wash hands thoroughly with soapy water. ➫ Never mouth-pipette these products.

Appendix A ➫ Contact the person responsible.

A1.3 Radioactive Risks Radioactive hazards are of different origins: • Gloves

➫ Change regularly to prevent contamination. Gloves are not a barrier to radiation. ➫ Regularly check surfaces, screens, hands 35 33 and equipment for contamination ( S, P). ➫ Store in containers stored in a special room. ➫ It is necessary to be familiar with safety precautions.

• Protection/control • Storage of radioactive sources • Radioprotection training courses

Table A1

Isotopes 35

S

33

P

Summary Table of Precautions to Be Taken during the Use of Radioisotopes

Wear a film badge No

Special equipment • Screens:

Risks, controls • No irradiation

— 0.2 mm glass, or — 0.3 mm Plexiglas • Two pairs of gloves to be worn

❑ Precautions Soak contaminated material in a diluted solution of decontaminant, then wash in running water ❑ Control/Wastes After radioactive decay (>10 half-lives) waste can be disposed of as nonradioactive waste.

• Contamination

➫ Decontamination ➫ Disposal must follow Health and Safety protocols.

A2 STERILIZATION The sterilization of solutions and of small equipment is indispensable for in situ PCR/RT-PCR.  Equipment • Aluminum foil • Autoclave • Autoclave tape • Oven ❑ Minor equipment • Glass staining trays

➫ Maintenance of clean conditions and the use of solutions exclusively for in situ PCR/RT-PCR

➫ The surface pattern changes after sterilization. ➫ Glass equipment is autoclaved or sterilized in an oven at 50°C.

351

Equipment ❑ Protocol Treatment of equipment, including magnetic bars, in: • Oven • Autoclave

2h 180°°C 2h 105°°C or 30 min 125°°C

➫ RNases are almost completely destroyed by this treatment. ➫ Two bars. Allow the equipment to cool in the oven (risk of breakage if it is placed on a cold surface).

A3 PRETREATMENT OF SLIDES ❑ Equipment • Metal forceps • Oven or autoclave • Trays, slide holders • Slides ❑ Reagents • Acetone • Alcohol 95% • 3-Amino-propyl-tri-ethoxy-silane • Hydrochloric acid 10 N • Sterile distilled water ❑ Solutions • Cleaning: Alcohol/HCl (5 ml HCl for 1 l 95% alcohol) • Treatment: 3-Amino-propyl-tri-ethoxy-silane at 2% in acetone ❑ Precaution Gloves should be worn throughout slide preparation. ❑ Protocol a. Wash: • Alcohol/HCl Overnight • Running water 1h • Distilled water 1 min b. Dry the slides in oven. 180°°C 60 min c. Allow to cool. d. Immerse in treatment solution. 5–15 s e. Wash: • Acetone 2 × 1 min • Sterile water 1 min f. Dry. Overnight 42°°C ❑ Storage rt Up to a year 352

➫ See Appendix B1.1. ➫ Stable solution, which can be stored ➫ Solution unstable; prepare just before use.

➫ Sterilization is necessary.

➫ Room temperature ➫ Dust-free conditions

Appendix A

A4

ELECTROPHORESIS OF PCR PRODUCTS IN AGAROSE GEL

❑ Equipment • Electrophoresis apparatus — Gel former tray — Combs — Tank ❑ Reagents • TAE buffer — Dissolve 242 g Tris base and 57.1 ml of glacial acetic acid in a final volume of 900 ml of water — Add 100 ml of 0.5 M EDTA; pH 8.0 • Ethidium bromide • Agarose for gel electrophoresis • 6X DNA loading buffer: — 30% (v/v) glycerol — 0.25% (w/v) bromophenol blue in TE buffer pH 7.4 • Ultraviolet transilluminator • DNA size marker ❑ Protocol a. Weigh the appropriate amount of agarose and dissolve it in 100 ml of TAE buffer by boiling.

b. Allow the agarose to cool to about 60°C and add 5 µl of ethidium bromide stock solution for 100 ml of agarose. c. Allow any bubbles to disperse and pour the agarose into a gel former tray appropriate for the electrophoresis tank to be used, inserting combs to cast wells allowing for 20 to 25 µl sample volume. d. Let the agarose solidify. 30 min rt e. Place the gel in the electrophoresis tank. f. Add sufficient TAE buffer to cover the electrodes and the gel. g. Mix 3 µl of 6X loading buffer with 15 µl of PCR product. h. Mix 3 µl of 6X loading buffer with 15 µl of size marker. i. Add the size marker to one lane of the gel and each sample to the other lanes.

➫ Stock solution: 50X

➫ Stock solution: 10 mg/ml in water ➫ Or Orange G if the expected product is <300 bp ➫ Or TAE buffer ➫ Diluted to 50 ng/µl in TE buffer (see Appendix B3.6) ➫ This can be done in a microwave oven. ➫ For PCR products of 300 to 1500 bp prepare 1.5% (w/v) agarose and for products <300 bp prepare 2.0% agarose. ➫ Wear protective gloves when handling ethidium bromide or gel containing it.

➫ Carefully pipette the sample into the well, below the surface of the TAE buffer. 353

Equipment j. Electrophorese the 30–45 min samples until the dye 80–100 V front has moved 3 to 4 cm 60–70 mA down the gel. k. Examine the gel on an ultraviolet transilluminator and compare the PCR product band with the size marker to determine the size of the amplified product.

354

Appendix B REAGENTS B1 WATER B1.1

 Reagents • Distilled water  Precaution Do not keep after opening.  Protocol Sterilize in an autoclave.  Storage

B1.2

➫ Sufficient for the detection of doublestranded DNA (e.g., viruses)

Sterile

2h 105°°C At rt

Diethylpyrocarbonate (DEPC)

 Reagents/Solutions • DEPC • Distilled water  Precaution DEPC is dangerous.  Protocol a. Mix: • DEPC • Water b. Shake under a fume hood. c. Sterilize in an autoclave.  Storage

➫ Some weeks ➫ Indispensable ➫ C6H10O5

0.5–1 ml 1000 ml Overnight 30 min 105°°C At rt

➫ Mw = 162.1

➫ Some weeks

B2 SOLUTIONS B2.1

Agarose

 Products/Solutions • Agarose • Fixation buffer  Precaution Cover the container when the agarose is being dissolved to avoid evaporation.

➫ Solution stock: 2% ➫ Molecular-biology quality ➫ PBS buffer, phosphate, or cacodylate ➫ See Appendix B3.2 and B3.4.

355

Reagents  Protocol a. Dissolve the agarose in the fixation buffer: • Agarose 2g • Fixation buffer 100 ml b. After dissolving the agarose keep the solution in an oven.  Storage • Oven 56°°C

B2.2

 Reagents/Solutions • Ammonium acetate • 10 N hydrochloric acid • Sterile water  Precautions Check that the powder is dry—volatile reagent.  Protocol • Ammonium acetate 57.81 g • Sterile water 80 ml a. Mix. b. Adjust the pH to 5.5 with HCl. • Sterile water to 100 ml  Sterilization Sterilize in an autoclave. 2h 105°°C  Storage • At −20°C in aliquots of 50 to 100 µl • At room temperature for some months

Calcium Chloride

B2.3.1

Calcium chloride

 Storage 356

➫ CH3 CO2NH4 ➫ See Appendix B1.1.

➫ Mw = 77.08

➫ Stock solution: 1 M

 Reagents/Solutions • Calcium chloride • Sterile water  Precaution None  Protocol • Calcium chloride • Sterile water  Sterilization Sterilize in an autoclave.

➫ Keep at this temperature until use (some hours). ➫ Stock solution: 7.5 M

Ammonium Acetate

B2.3

➫ Use a water bath.

➫ AR quality, to be used only for in situ PCR/ RT-PCR ➫ CaCl2·2H2O ➫ See Appendix B1.1.

14.7 g to 100 ml 2h 105°°C At rt

➫ Mw = 147.02

➫ Some weeks before opening

Appendix B B2.3.2 Calcium chloride/cobalt chloride  Reagents/Solutions • Calcium chloride • Cobalt chloride • Sterile water  Precaution None  Protocol • Calcium chloride 0.029 g • Cobalt chloride 0.025 g • Sterile water to 100 ml  Sterilization Sterilize in an autoclave. 2h 105°°C  Storage At rt

B2.4

➫ CaCl2·2H2O ➫ CoCl2 ➫ See Appendix B1.1.

➫ Mw = 147.02 ➫ Mw = 129.83

➫ Some weeks before opening

Deionized Formamide

 Equipment • Sterile flask • Whatman filter N° 1M  Reagents/Solutions • Amberlite resin, 20 to 50 mesh • Formamide  Precaution Avoid contact.  Protocol • Amberlite 5g • Formamide filter 50 ml a. Shake gently. 30 min b. Filter on Whatman paper.  Storage At −20°C in sterile 1 ml tubes, and store in lightfree conditions.

B2.5

➫ Stock solution: 2 mM CaCl2 /2 mM CoCl2

➫ CH3NO ➫ Commercially available solution ➫ Mw = 45.04

➫ Do not thaw. If it is liquid at −20°C, it must not be used. ➫ Stock solution: 50X

Denhardt’s Solution

 Reagents/Solutions • Bovine serum albumin (fraction V) • Ficoll 400 • Polyvinylpyrolidone • Sterile water  Precaution Risk of bacterial contamination  Protocol a. Add: • BSA • Ficoll 400

➫ Molecular-biology quality

➫ BSA, crystallized five times ➫ Synthetic polymer of sucrose ➫ See Appendix B1.1.

1g 1g

357

Reagents • Polyvinylpyrolidone 1g • Sterile water to 100 ml b. Leave the mixture to hydrate overnight before shaking. c. Shake the mixture gently and intermittently for some days.  Storage In aliquots. At −20°°C

➫ Can be thawed and refrozen

B2.6

➫ Stock solution: 50%

Dextran Sulfate

 Reagents/Solutions • Dextran sulfate • Sterile water  Precaution Reagent very difficult to pipette. Prepare extemporaneously only the quantity necessary.  Protocol a. Add to a sterile Eppendorf tube: • Dextran sulfate 50 mg • Sterile water 65 µl b. Mix. c. Microcentrifuge for a few seconds.  Storage is possible In aliquots of 100 µl At −20°°C

➫ Molecular-biology quality

B2.7

➫ Stock solution: 1 M

Dithiothreitol (DTT)

 Reagents/Solutions • DTT • Sterile water  Precaution Very volatile reagent, unstable at room temperature Does not resist denaturation  Protocol a. Add to a sterile Eppendorf tube: • Dithiothreitol 1.54 g • Sterile water 10 ml b. Mix.  Storage In aliquots of 100 µl At −20°°C

358

➫ See Appendix B1.1.

➫ Mw = 500.00

➫ It is recommended that it be stored in aliquots as the other components of the PCR/RT-PCR mixture should be.

➫ Molecular-biology quality ➫ C4H10O2S2 ➫ See Appendix B1.1. ➫ Denatured quickly in the PCR/RT-PCR buffer ➫ Mw = 154.24

➫ Must never be refrozen. The strong smell indicates that the product is in good condition.

Appendix B

B2.8

 Reagents/Solutions • Herring sperm DNA • Salmon sperm DNA • Sterile water  Precaution Risk of bacterial contamination  Protocol • DNA • Sterile water Sonicate for 10 min  Storage

B2.9

➫ Use commercially available preparations. ➫ See Appendix B1.1.

10 mg to 1 ml At −20°°C

➫ Maximum power ➫ May be thawed and refrozen ➫ Stock solution: 1 mg/ml

DNase I

 Reagents/Solutions • DNase I • Sterile water  Precaution Risk of RNase contamination  Protocol Dissolve • DNase I • Sterile water  Storage In aliquots of 100 µl

B2.10

➫ Stock solution: 10 mg/ml sterile water

DNA

➫ Molecular-biology quality ➫ Check the activity of the enzyme. ➫ See Appendix B1.1.

1 mg 1 ml

➫ To be expressed in units

At –20°°C

Ethylene Diamine Tetra-Acetic Acid (EDTA)

 Reagents/Solutions • EDTA • 10 N sodium hydroxide • Sterile water  Precaution Toxic reagent  Protocol a. Dissolve • EDTA 186 g • Sterile water to 1 l b. Adjust the pH to 8.0 with sodium hydroxide.  Sterilization Sterilize in an autoclave. 2h 105°°C  Storage In aliquots At rt

➫ Stock solution: 500 mM ➫ RP quality, to be used only for in situ PCR/ RT-PCR ➫ C10H14N2O8Na2·2H2O or Titriplex III ➫ See Appendix B1.1.

➫ Mw = 372.24

359

Reagents

B2.11

➫ Stock solution: 4 M

Lithium Chloride

 Reagents/Solutions • Lithium chloride • Sterile water  Precaution Risk of bacterial contamination  Protocol • Lithium chloride • Sterile water a. Mix b. Filter on a 0.22 µm filter.  Storage

B2.12

➫ RP quality, to be used only for in situ PCR/ RT-PCR ➫ LiCl ➫ See Appendix B1.1.

8.48 g to 50 ml At 4°°C

 Reagents/Solutions

 Storage

B2.13

B2.14

Proteinase

B2.14.1

Proteinase K

 Reagents/Solutions • Proteinase K • Sterile water

360

➫ RP quality, to be used only for in situ PCR/ RT-PCR ➫ MgCl2·6H2O ➫ See Appendix B1.1.

20.3 g to 100 ml

➫ Mw = 203.30

2h 105°°C At rt ➫ Stock solution: 10 mg/ml

Poly (A)

 Reagents/Solutions • DEPC-treated water • Poly (A)  Precaution Risk of hydrolysis by RNase  Storage

➫ For a few weeks ➫ Stock solution: 1 M

Magnesium Chloride

• Magnesium chloride • Sterile water  Precaution None  Protocol • Magnesium chloride • Sterile water  Sterilization Sterilize in an autoclave.

➫ Mw = 42.39

➫ See Appendix B1.2. ➫ C11H17N4O12 (polyribonucleotide adenylate) At –20°°C

➫ Some weeks

➫ Stock solution: 10 mg/ml

➫ See Appendix B1.1.

Appendix B  Precaution Weak proteolysis at room temperature  Protocol a. Place in a sterile Eppendorf tube: • Proteinase K 10 mg • Sterile water 1 ml b. Mix.  Storage In aliquots of 100 µl At –20°°C

B2.14.2 Pepsine  Reagents/Solutions • Pepsine • Sterile water  Precaution Weak proteolysis at room temperature  Protocol a. Place in a sterile Eppendorf tube: • Pepsine 10 mg • Sterile water 1 ml b. Mix  Storage In aliquots of 100 µl At –20°°C

B2.14.3 Pronase  Reagents/Solutions • Pronase • Sterile water  Precaution Weak proteolysis at room temperature  Protocol a. Place in a sterile Eppendorf tube: • Pronase 10 mg • Sterile water 1 ml b. Mix  Storage In aliquots of 100 µl At −20°°C

B2.15

RNA

 Reagents/Solutions • DEPC-treated water • Yeast tRNA

➫ Use commercially available solutions.

➫ Dilute in Tris–HCl/CaCl2 buffer (see Appendix B3.7.2). ➫ Stock solution: 10 mg/ml

➫ See Appendix B1.1. ➫ Use commercially available solutions.

➫ Dilute in Tris/EDTA buffer 1X (see Appendix B3.6.1). ➫ Stock solution: 10 mg/ml

➫ See Appendix B1.1. ➫ Use commercially available solutions.

➫ Dilute in Tris/EDTA buffer 1X (see Appendix B3.6.1). ➫ Stock solution: 10 mg/ml sterile water ➫ See Appendix B1.2.

361

Reagents  Precaution Risk of hydrolysis  Protocol • RNA • DEPC-treated water Sonicate 10 min.  Storage

B2.16

➫ ➫ ➫ ➫

At −20°°C ➫ Solution stock: 20% buffered ➫ See Appendix B3.4. ➫ 0.2% Saponin may be used.

20 ml 80 ml At 4°°C

➫ For some weeks ➫ Stock solution: 3 M

 Reagents/Solutions

362

Molecular-biology quality Bovine pancreas ribonuclease See Appendix B1.1. See Appendix B3.6.

100 µg 1 ml

Sodium Acetate

• Glacial acetic acid • Sodium acetate • Sterile water  Precaution None  Protocol • Sodium acetate • Sterile water

➫ Maximum power ➫ May be thawed and refrozen ➫ Stock solution: 100 µg/ml

Sarcosyl

 Reagents • Phosphate buffer or PBS • Sarcosyl  Precaution Toxic  Protocol • Sarcosyl • Sterile water  Storage

B2.18

At –20°°C

RNase A

 Reagents/Solutions • RNase A • Sterile water • TE/NaCl buffer  Precaution Risk of DNase contamination.  Protocol • RNase A • Sterile water  Storage ➫ In aliquots of 40 µl

B2.17

10 mg to 1 ml

➫ AR quality, to be used only for in situ PCR/RT-PCR ➫ CH3 CO2H ➫ CH3 COONa ➫ See Appendix B1.1.

24.6 g 80 ml

➫ Mw = 82.03

Appendix B a. Mix with magnetic stirring. b. Adjust the pH to 4.8 with acetic acid. • Sterile water to 100 ml  Sterilization Sterilize in an autoclave. 2h 105°°C  Storage In aliquots of 50 to 100 µl. At rt

➫ For some months

B2.19

➫ Stock solution: 5 M

Sodium Chloride

 Reagents/Solutions • Sodium chloride • Sterile water  Precaution None  Protocol • Sodium chloride • Sterile water  Sterilization Sterilize in an autoclave.  Storage

B2.20

14.6 g to 50 ml 2h 105°°C At rt

➫ Mw = 58.44

➫ For some months ➫ Stock solution: 10 N

Sodium Hydroxide

 Reagents/Solutions • Sodium hydroxide • Sterile water  Precaution Extremely corrosive reagent  Protocol • Sodium hydroxide • Sterile water Mix slowly.  Storage

B2.21

➫ AR or molecular-biology quality, to be used only for in situ PCR/RT-PCR ➫ NaCl ➫ See Appendix B1.1.

➫ RP quality ➫ NaOH ➫ See Appendix B1.1.

40 g to 100 ml

Tris

 Reagents/Solutions • 10 N hydrochloric acid • Sterile water • Tris–hydroxymethyl–aminomethane  Precaution None

➫ Mw = 40.00

At rt ➫ Stock solution: 1 M

➫ See Appendix B1.1. ➫ C4H11NO3, Tris base in powder

363

Reagents  Protocol Dissolve: • Tris base • Sterile water  Storage

B2.22

0.1–0.3 g

• Sterile water  Storage

to 100 ml At rt

B3.1

➫ Some weeks

➫ AR quality ➫ See Appendix B1.1. ➫ C34H62O11

➫ Mw = 646.87 ➫ Difficult to pipette with exactitude ➫ Some weeks

BUFFERS ➫ 100 mM Triethanolamine buffer; pH 8.0

Acetylation Buffer

 Reagents/Solutions • 10 N hydrochloric acid • Sterile water • Triethanolamine  Precaution Toxic  Protocol a. Mix: • Triethanolamine • Sterile water b. Adjust pH: • HCl • Sterile water  Storage

B3.2

➫ Mw = 121.16

➫ Stock solution: 0.1 to 0.3%

Triton X-100

 Reagents/Solutions • Sterile water • Triton X-100  Precaution Reagent difficult to pipette  Protocol Dissolve: • Triton X-100

B3

Cacodylate Buffer

 Products/Solutions • N hydrochloric acid • Sodium cacodylate • Sterile water  Precaution None 364

121 g to 1 l At rt

➫ RP quality ➫ See Appendix B1.1. ➫ C6H15NO3 (Tris (hydroxy-2-ethyl)-amine)

3.32 ml 200 ml pH 8.0 ≈1 ml to 250 ml At 4°°C

➫ Mw = 149.19

➫ Some hours ➫ 200 mM Cacodylate buffer; pH 7.4 ➫ AR quality ➫ Na(CH3)2AsO2·3H2O ➫ See Appendix B1.1.

Appendix B  Protocol a. Dissolve: • Sodium cacodylate • Sterile water b. Check the pH.  Sterilization Autoclave.  Storage

B3.3

4.28 g to 100 ml 7.4 2h 105°°C At 4°°C

DNase I Buffer

 Reagents/Solutions • Bovine serum albumin • 10 N hydrochloric acid • 500 mM magnesium chloride • Sterile water • 1 M Tris  Precaution Risk of RNase contamination; prepare under sterile conditions  Protocol • Tris–HCl; pH 7.6 500 mM • MgCl2 100 mM • BSA 0.5 mg/ml  Storage In aliquots At –20°°C

B3.4

Phosphate Buffer

B3.4.1

1 M Phosphate

 Reagents/Solutions • Disodium phosphate • Monosodium phosphate • Sterile water  Precaution Preparations obtained in sachets should be transferred to sterile containers, with sterile water.  Protocol a. Dissolve: • Disodium phosphate 14.19 g • Monosodium phosphate 13.8 g • Sterile water to 100 ml b. Check pH. pH 7.4  Sterilization Autoclave. 2h 105°°C  Storage At rt or 4°°C

➫ Mw = 214.05

➫ Some days ➫ Stock solution: 10X ➫ RP quality ➫ BSA 5X, crystallized ➫ See Appendix B2.12. ➫ See Appendix B1.1. ➫ See Appendix B2.21.

➫ See Appendix B3.7.1.

➫ Some hours

➫ Stock solution ➫ AR quality ➫ Na2HPO4. Hydrated reagents can be used, taking into account variations in molar mass. ➫ NaH2PO4·H2O ➫ See Appendix B1.1.

➫ Mw = 141.96 ➫ Mw = 138.00

➫ Some weeks 365

Reagents B3.4.2

 Products/Solutions • 1 M phosphate buffer • 5 M sodium chloride • Sterile water  Precaution None  Protocol a. Add: • 5 M sodium chloride • 1 M phosphate buffer • Sterile water b. Check pH.  Sterilization Autoclave.  Storage B3.4.3

➫ 100 mM phosphate buffer/300 mM NaCl; pH 7.4

Phosphate/NaCl

➫ ➫ ➫ ➫

6 ml 10 ml to 100 ml pH 7.4 2h 105°°C At rt or 4°°C

PBS

 Reagents/Solutions • • • •

Disodium phosphate Monopotassium phosphate Potassium chloride Sodium chloride

• 10 N sodium hydroxide • Sterile water  Precaution Risks of contamination  Protocol a. Add: • Sodium chloride 8.76 g • Potassium chloride 0.74 g • Disodium phosphate 3.58 g • Monopotassium phosphate 1.90 g • Sterile water to 1 l b. Adjust the pH with sodium hydroxide. 7.4  Sterilization Sterilize in an autoclave. 2h 105°°C  Storage At rt up to opening then at 4°°C

366

AR quality See Appendix B3.4.1. See Appendix B2.19. See Appendix B1.1.

➫ Some days ➫ Phosphate buffer saline ➫ 10 mM mono-di-phosphate buffer/150 mM NaCl/10 mM KCl; pH 7.4 ➫ Can be prepared in stock solution 10X ➫ AR quality, and to be used only for in situ PCR/RT-PCR ➫ Na2HPO4·12H2O ➫ KH2PO4·3H2O ➫ KCl ➫ NaCl ➫ See Appendix B2.19. ➫ See Appendix B2.20. ➫ See Appendix B1.1.

➫ ➫ ➫ ➫

Mw = 58.44 Mw = 74.56 Mw = 358.14 Mw = 190.15

➫ Some weeks ➫ Some hours

Appendix B

B3.5

➫ Standard Saline Citrate buffer ➫ Stock solution: 20X

SSC Buffer

 Reagents/Solutions • 10 N sodium hydroxide • Sodium chloride • Sodium citrate • Sterile water  Precaution None  Protocol a. Add: • Sodium chloride • Sodium citrate • Sterile water b. Adjust the pH with sodium hydroxide.  Sterilization Autoclave.  Storage

B3.6

➫ 3 M sodium chloride; 300 mM sodium citrate; pH 7.0 ➫ RP quality ➫ See Appendix B2.20. ➫ NaCl ➫ C6H5Na3O7·2H2O ➫ See Appendix B1.1.

175.3 g 88.2 g to 1 l 7.0

2h 105°°C At rt prior to opening then at 4°°C

TE (Tris–EDTA) Buffer

 Reagents • EDTA • Tris (hydroxymethyl–aminomethane) B3.6.1

TE buffer

 Reagents/Solutions • EDTA • 10 N hydrochloric acid • Sterile water • Tris

➫ Mw = 58.44 ➫ Mw = 294.10

➫ Some weeks ➫ Some hours ➫ Tris/EDTA ➫ AR quality, to be used only for in situ PCR/RT-PCR ➫ C10H14N2O8Na2⋅2H2O (ethylene diamine tetra-acetic acid, or Titriplex III) ➫ C4H11NO3 ➫ Stock solution: 10X ➫ 100 mM Tris–HCl/10 mM EDTA; pH 7.6 ➫ C10H14N2O8Na2·2H2O or 500 mM stock solution (see Appendix B2.13) ➫ See Appendix B1.1. ➫ C4H11NO3 or 1 M stock solution (see Appendix B2.22)

 Precaution Toxicity of EDTA

367

Reagents  Protocol a. Add: • Tris • EDTA b. Adjust the pH with HCl. • Sterile water  Sterilization Sterilize in an autoclave.  Storage

B3.6.2

12.1 g 3.7 g 7.6 to 1 l

2h 105°°C At rt up to opening then at 4°°C

 Storage

➫ AR quality ➫ NaCl ➫ See Appendix B1.1. ➫ See Appendix B3.6.1.

29.2 g to 100 ml 7.6 2h 105°°C At rt until opened then at 4°°C

B3.7

Tris–HCl Buffer

B3.7.1

Tris–HCl buffer

 Reagents/Solutions • 10 N hydrochloric acid • Sterile water • 1 M Tris  Precaution Risk of contamination by exogenous organisms  Protocol a. Add: • Tris 1 vol • Sterile water 9 vol

368

➫ Some weeks ➫ Some days ➫ Stock solution: 10X ➫ 100 mM Tris–HCl/10 mM EDTA/5 M NaCl; pH 7.6

TE–NaCl buffer

 Products/Solutions • 10 N hydrochloric acid • Sodium chloride • Sterile water • 10X TE buffer  Precaution Toxicity of EDTA  Protocol a. Add: • Sodium chloride • 10X TE buffer b. Adjust the pH with HCl.  Sterilization Autoclave.

➫ Mw = 121.16 ➫ Mw = 372.24

➫ Mw = 58.44

➫ Some weeks ➫ Some hours

➫ Solution: 100 mM ➫ RP quality ➫ See Appendix B1.1. ➫ See Appendix B2.21.

Appendix B b. Adjust the pH with HCl: • pH • pH  Sterilization  Storage B3.7.2

Tris–HCl/MgCl2 buffer

 Reagents/Solutions • 10 N hydrochloric acid • 1 M magnesium chloride • Sterile water • 1 M Tris

➫ Very difficult, if not impossible ➫ Some days ➫ 20 mM Tris–HCl/2 mM CaCl2; pH 7.6 ➫ RP quality ➫ See Appendix B2.3. ➫ See Appendix B1.1. ➫ See Appendix B2.21.

20 ml 2 ml 7.6 to 1 l At 4°°C

➫ Very difficult, if not impossible ➫ Some days ➫ 50 mM Tris–HCl/50 mM glycine; pH 7.4

Tris–HCl/glycine buffer

 Reagents/Solutions • Glycine • 10 N hydrochloric acid • Sterile water • 1 M Tris  Precaution Risk of bacterial contamination  Protocol a. Add: • Glycine • Tris • Sterile water b. Adjust the pH with HCl  Sterilization  Storage In aliquots

B3.7.4

At 4°°C

Tris–HCl/CaCl2 buffer

 Reagents/Solutions • 1 M calcium chloride • 10 N hydrochloric acid • Sterile water • 1 M Tris  Precaution Risk of contamination  Protocol a. Add: • Tris • Calcium chloride b. Adjust the pH with HCl: • Sterile water  Sterilization  Storage B3.7.3

7.6 8.5

➫ RP quality ➫ C2H5NO2 ➫ See Appendix B1.1. ➫ See Appendix B2.21.

0.37 g 5 ml to 100 ml 7.4

➫ Mw = 75.07

➫ Very difficult, if not impossible At –20°°C At 4°°C

➫ Some weeks ➫ Some hours ➫ 10 mM Tris–HCl/5 mM MgCl2; pH 7.3 (dilution buffer for DNase) ➫ RP quality ➫ See Appendix B2.12. ➫ See Appendix B1.1. ➫ See Appendix B2.21. 369

Reagents  Precaution Risk of bacterial contamination  Protocol a. Add: • 1 M Tris • 1 M MgCl2 b. Adjust the pH with HCl: • Sterile water  Storage In aliquots

B3.7.5

➫ Some weeks ➫ Some hours ➫ 20 mM Tris–HCl/300 mM NaCl; pH 7.6 ➫ Visualization buffer ➫ RP quality ➫ See Appendix B2.19. ➫ See Appendix B1.1. ➫ See Appendix B2.21.

6 ml 2 ml to 100 ml 7.6

➫ 20 mM Tris–HCl/300 mM NaCl/50 mM MgCl2

Tris–HCl/NaCl/MgCl2 buffer

 Reagents/Solutions • 10 N hydrochloric acid • 1 M magnesium chloride • 5 M sodium chloride • Sterile water • 1 M Tris  Precaution Risk of bacterial contamination  Protocol a. Add: • 1 M Tris • 5 M NaCl • 1 M MgCl2 b. Adjust the pH with HCl: • pH • pH • Sterile water 370

At –20°°C At 4°°C

Tris–HCl/NaCl buffer

 Reagents/Solutions • 10 N hydrochloric acid • 5 M sodium chloride • Sterile water • 1 M Tris  Precaution Risk of bacterial contamination  Protocol a. Add: • 5 M sodium chloride • 1 M Tris • Sterile water b. Adjust the pH with HCl.  Storage Prepare just before use. B3.7.6

1 ml 500 µl 7.3 to 100 ml

➫ RP quality ➫ ➫ ➫ ➫

See Appendix B2.12. See Appendix B2.19. See Appendix B1.1. See Appendix B2.21.

20 ml 60 ml 50 ml 7.6 9.5 to 1 l

➫ Visualization of peroxidase ➫ Visualization of alkaline phosphatase

Appendix B  Storage Prepare extemporaneously.

B4 FIXATIVES B4.1

Formol

B4.1.1

Neutral buffered formalin

 Reagents/Solutions • Na2HPO4 • NaH2PO4H2O • Formaldehyde 37 to 40% • Distilled water  Precaution Risks for the eyes and the respiratory passages  Protocol a. Dissolve • Na2HPO4 6.5 g • NaH2PO4 H2O 4g b. Add: Distilled water and 100 ml 500 ml 40% formaldehyde and make up to 1 l with distilled water B4.1.2

➫ Commercial solution ➫ DEPC water (see Appendix B2.1.2) ➫ Manipulation under a hood

➫ Check the pH and adjust if necessary to pH 7.2 using appropriate salt

Formol saline

 Reagents/Solutions • Formaldehyde 37 to 40% • Distilled water • Sodium chloride  Precaution Risks for the eyes and the respiratory passages  Protocol a. Mix 100 ml formaldehyde and 900 ml of distilled water. b. Dissolve 9 g of sodium chloride in the mixture.  Storage To be avoided after dilution

B4.2

➫ Solution stock

Glutaraldehyde (2.5%)

 Products/Solutions • 25 to 70% glutaraldehyde

• 200 mM phosphate buffer

➫ Commercial solution ➫ DEPC H2O (see Appendix B2.1.2) ➫ See Appendix B2.19. ➫ Manipulation under a hood

➫ Solution to be prepared just before use ➫ Electron microscopy quality ➫ OCH(CH2)3CHO ➫ The higher the concentration, the stabler the solution ➫ See Appendix B3.4. 371

Reagents  Precaution Volatile and dangerous chemical  Protocol Mix: • 25% glutaraldehyde • Phosphate buffer • Distilled water  Storage Use immediately after preparation.

B4.3

➫ Use under a fume hood

1 ml 5 ml to 10 ml

• 1 M phosphate buffer • 10 N sodium hydroxide Paraformaldehyde 40%

 Precaution Manipulate under hood.  Protocol a. Place in an Erlenmeyer: • Distilled water 100 ml • Paraformaldehyde 80 g b. Allow to hydrate for some minutes, then mix the solution with magnetic stirring. c. Add caustic soda until the solution clears: ≈ 0.5 ml • NaOH • Distilled water to 200 ml d. Close the recipient with aluminum foil. e. Heat and shake. 20 min 60°°C f. Filter and allow to cool.  Storage In aliquots At 4°C At −20°°C

B4.3.2

Paraformaldehyde 4%

 Precaution Manipulation under fume hood

372

➫ A couple of hours at 4°C

Paraformaldehyde

 Equipment • Hood • Magnetic stirring, with heating  Reagents/Solutions • Distilled water • Paraformaldehyde in powder

B4.3.1

➫ Depending on the concentration of the original solution

➫ ➫ ➫ ➫ ➫ ➫

RP quality See Appendix B1.1. HO(CH2O)nH In the form of nonhydrated powder See Appendix B3.4.1. See Appendix B2.20.

➫ Stock solution

➫ The solution is milky in appearance.

➫ The solution clears.

➫ 1 month ➫ Several months ➫ Do not refreeze

Appendix B  Protocol a. Place in a glass flask: • Paraformaldehyde • Distilled water b. Stir while heating. c. Allow to cool, and add: • 1 M phosphate buffer d. Adjust the pH with NaOH • Distilled water e. Filter and allow to cool.  Storage To be used immediately

B4.3.3

4g 50 ml 60°°C 10 ml pH 7.4 to 100 ml

➫ See Appendix B3.4.1.

At –20°°C

➫ Some days ➫ Do not refreeze.

Paraformaldehyde 4%/ glutaraldehyde 0.05%

 Reagents/Solutions • Glutaraldehyde 25 to 75% • 4% Paraformaldehyde (PF) in 100 mM phosphate buffer; pH 7.4  Precaution Manipulation under hood  Protocol • 4% buffered paraformaldehyde 200 ml • Glutaraldehyde 133–400 µl

➫ Working solution

➫ OCH(CH2)3CHO, microscopy quality; the higher the concentration, the stabler the solution. ➫ See Appendix B4.3.2.

➫ According to the glutaraldehyde concentration

 Storage Use immediately.

B5 EMBEDDING MEDIA B5.1 • • • • • • •

Materials

Disposable pipettes Fume hood Glass containers Gloves Magnetic stirrer Mask Propipette

➫ Or electric stirrer

373

Reagents

B5.2

Epoxy Resins

B5.2.1

Epon–araldite

 Products/Solutions • Araldite 502 • Dodecenyl succinic anhydride • Hexahydrophtalic anhydride • 2,4,6-Tris-dimethylamine methyl phenol  Precautions Toxic products

➫ Risk of developing allergies ➫ Wear a mask. ➫ Wear gloves. All traces of resin on the skin must be removed with soapy water.

Avoid all contact with the skin.  Protocol a. Mix all the following products in a glass container: • Araldite 502 20 ml • DDSA 60 ml • Epox 812 25 ml b. Add the hardener to the mixture: • Epon–araldite mixture 20 ml • DMP 30

➫ DDSA, hardener ➫ Epox 812 resin ➫ DMP 30 (accelerator); very dangerous and irritant chemical

300 µl

➫ The mixture is very viscous.

➫ 300 µl of resin is required to fill a small gelatin capsule (size 00). ➫ The amount of the product required can rise by 20% depending on how often the container is opened.

 Storage • Prepare just before use and keep away from light. B5.2.2

Epon

 Products/Solutions • Dodecenyl succinic anhydride • Epox 812 resin • Methyl nadic anhydride • 2,4,6-Tris-dimethylamine methyl phenol  Precautions Toxic products Avoid all skin contact  Protocol ❶ Stock solution a. Thoroughly mix all the following products in a glass container:

374

➫ DDSA, hardener ➫ Aliphatic carbon chain (mixture of glycidyl ethers and mono-trisubstituted glycerol) ➫ MNA, hardener ➫ DMP 30, accelerator; very dangerous and irritant chemical ➫ Risk of developing allergies ➫ Wear a mask. ➫ Gloves must be worn. ➫ The mixture is very viscous. A glass rod fixed to an electric mixer may be used to prepare a large quantity.

Appendix B • DDSA • Epox 812 • MNA ❷ Solution for use b. Add the hardener to the mixture: • Epon mixture • DMP 30

50 ml 81 ml 44.5 ml ➫ To polymerize the resin 20 ml 300 µl

 Storage • The mixture without the hardener can be kept at room temperature for 1 month. • The mixture with the hardener can be kept at 4°C for several hours or overnight.

➫ The amount of the product required can rise by 20% depending on how often the container is opened.

➫ Time taken to impregnate the tissue.

B5.3

Acrylic Resins

➫ Methacrylate resin

B5.3.1

Lowicryl K4M

➫ 2-hydroxyethyl-acrylate; resin sold as a kit

 Products/Solutions • Lowicryl K4M • Solution A (Cross-linker) • Solution B (monomer) • Initiator C  Precautions Toxic products Very toxic resin Avoid all contact with the skin Avoid inhaling the vapors

 Protocol

a. Thoroughly mix the following products in a glass container: • Initiator C 0.1 g • Solution B 17.30 ml b. Add • Solution A 2.70 ml c. Embed the samples.  Storage • The different products of the kit are kept at rt in a dry, dust-free place away from heat. • The mixture can be kept for up to 2 days at −30°C.

➫ Clear liquid, distinctive smell ➫ Accelerates polymerization ➫ Risk of developing allergies ➫ An automatic embedding system is available. ➫ Methacrylate is irritant to the skin, eyes and respiratory system. Gloves and a mask must be worn. All traces of the resin on the skin must be removed with running water. ➫ Pipette using a propipette. ➫ Use a fume hood. ➫ Quantity to prepare: • 1 ml/tube for impregnation • 500 µl/gelatin capsule for embedding ➫ The mixture is very runny. ➫ Wear a mask during weighing. ➫ Pipette using an automatic pipette. ➫ Stir gently (to avoid bubbles). ➫ 1 to 2 years ➫ The storage depends on the time taken to impregnate the samples. 375

Reagents  Wastes Keep waste in special containers.

B5.3.2

➫ Do not throw down the sink. ➫ May be destroyed by incineration. ➫ London Resin Medium hardness

LR White medium

 Products/Solutions • LR White medium

➫ Acrylic polyhydroxyaromatic resin ➫ A single stock solution of methacrylate

• Accelerator  Precautions Weakly toxic and irritant resin

➫ Made up solely of monomers used in medicine and dentistry ➫ Less allergenic than Lowicryl K4M ➫ Recommended to wear a mask and gloves; pipette with a propipette ➫ Use in a fume hood ➫ Quantity to make up: • 1 ml per tube for impregnation • 0.5 ml per gelatin capsule for embedding

Avoid contact with the skin Avoid inhaling vapors  Protocol

a. Take out the products just before making the mixture. b. Place in a glass container: • LR White 5 ml • Accelerator 5 µl c. Mix immediately. 1–2 min d. Embed the samples very quickly.  Storage

At 4°°C

 Wastes Keep the waste in glass containers. B5.3.3

Unicryl

 Product • Unicryl  Precautions Toxic resin Avoid contact with the skin. Avoid inhaling vapors.  Protocol

a. Take out the stock solution just before impregnation.

376

➫ Use in a fume hood. ➫ Use a propipette. ➫ ≈1 drop (optional). ➫ Very quickly with a vorex. ➫ Polymerization is very fast: less than 15 min with an accelerator, several hours without. ➫ For several months ➫ Very sensitive to the heat ➫ Do not throw down the sink. ➫ Stock solution ready to use ➫ The commercially available solution is ready to use. ➫ Always wear gloves. ➫ Always wear a mask; pipette using a propipette under a fume hood. ➫ Use a fume hood. ➫ Quantity to make up: • 1 ml per tube for impregnation • 0.5 ml per gelatin capsule for embedding

Appendix B b. Embed biological samples at low temperature: Samples are dehydrated progressively at low temperature (from 0°C down to −20°C) without a substitution stage. After the last change of solvent, the stock solution is directly impregnates at −20°C for several hours.  Storage At –30°°C At 4°°C

➫ Rapid protocol ➫ Easy to use and the resin is ready to use, cutting down the risk of handling ➫ For several months ➫ For 1 year

B6 REVELATION B6.1

Autoradiography

B6.1.1

Standard developer

 Reagents/Solutions • Hydroquinone • Metol • Potassium bromide • Sodium carbonate • Sodium sulfite anhydride • Sterile water  Precaution Toxic reagent  Working solution

a. Dissolve the following components: • Metol 0.5 g • Hydroquinone 6g • Sodium sulfite 50 g • Sodium carbonate 32 g • Potassium bromide 2g • Running water to 1 l b. Filter.  Storage At 4°°C  Protocol • Pure 4 min 17°°C • Diluted in water (1:1 v/v) 6 min 17°°C B6.1.2

➫ ➫ ➫ ➫ ➫ ➫ ➫

AR quality C6H4(OH)2 [OHC6H4NH(CH3)]·2H2SO4 KBr Na2CO3 Na2SO3 See Appendix B1.1.

➫ To be manipulated with gloves ➫ Preferably ready-to-use solutions recommended by the manufacturer of the emulsions and films

➫ Anhydride ➫ Anhydride

Fixative

 Reagents/Solutions • Distilled water • Sodium thiosulfate

➫ Na2S7O3

377

Reagents  Working solution • Sodium thiosulfate 30% • Distilled water  Protocol • Working solution  Storage Some weeks at room temperature

B6.2

Immunocytology

B6.2.1

Blocking solutions

60 g 200 ml 1–5 min

B6.2.1.1 NONSPECIFIC SITES  Reagents/Solutions • Blocking agents — Bovine serum albumin (BSA) — Dried skimmed milk — Fish gelatin — Goat serum — Ovalbumin — 0.01% Triton X-100 • Buffers — 100 mM phosphate buffer; pH 7.4 — 50 mM Tris–HCl buffer/300 mM NaCl; pH 7.6  Precaution Do not leave at room temperature.  Working solution Dilute: • Blocking agents 1–2% • Buffer to 100 ml  Storage At −20°°C B6.2.1.2 ENDOGENOUS ALKALINE PHOSPHATASES ❶ Levamisole  Reagents/Solutions • Levamisol (2,3,5,6-tetrahydro-6phenylimidazole) • 50 mM Tris–HCl buffer/100 mM NaCl/ 50 mM MgCl2; pH 9.5  Precaution None  1 M stock solution Dissolve: • 100 mM levamisol 24 mg • Revelation buffer 1 ml 378

➫ Mw = 158.00 ➫ Minimum volume for a staining tray

➫ See Appendix B3.4. ➫ See Appendix B3.7.5.

➫ Some weeks ➫ Endogenous enzyme activity may be inhibited by levamisole or by heat treatment.

➫ C11H12N2S (2,3,5,6-tétrahydro-6-phenylimidazole) ➫ See Appendix B3.7.6.

➫ Mw = 240.80

Appendix B  1 mM working solution • Stock solution • Revelation buffer  Storage Use immediately after preparation.

1 µl 990 µl

B6.2.1.3 ENDOGENOUS PEROXIDASES ❶ Hydrogen peroxide/buffer  Reagents/Solutions • 30% hydrogen peroxide • 50 mM Tris–HCl/300 mM NaCl; pH 7.6  Precaution Avoid contact with the skin.  Solution for use Mix: • Hydrogen peroxide • Buffer  Storage Use immediately after preparation. ❷ Hydrogen peroxide/methanol  Reagents/Solutions • 30% hydrogen peroxide • 100% methanol  Precaution Contact dangerous  Working solution Mix: • Hydrogen peroxide • Methanol  Storage Use immediately after preparation. B6.2.2

➫ 110 V ➫ See Appendix B3.7.5, or use a phosphate buffer (see Appendix B3.4). ➫ Wash in running water

3 ml 100 ml

➫ 110 V

3 ml 100 ml

Chromogens

B6.2.2.1 ALKALINE PHOSPHATASE ❶ NBT-BCIP  Reagents/Solutions • Dimethylformamide • Substrates — NBT (Nitroblue tetrazolium) — BCIP (5-bromo-4-chloro-3-indolyl phosphate) • Tris–HCl/NaCl/MgCl2 buffer; pH 9.5  Precaution Avoid contact with the skin. Use immediately after preparation and keep out of the light.

➫ There are other chromogen substrates that give different color precipitates (i.e., Fast-Red). ➫ Use a commercial solution. ➫ (CH3)2NOCH ➫ C40H30Cl2N10O6 ➫ C8H6NO4BrCIP⋅C7H9N ➫ See Appendix B3.7.6.

379

Reagents  Working solution Mix: • NBT

• BCIP

0.34 mg or 4.5 µl 0.18 mg or 3.5 µl to 1 ml

➫ Mw = 817.70 ➫ 75 mg/ml dimethylformamide ➫ Mw = 433.60 ➫ 50 mg/ml dimethylformamide

• Visualization buffer  Storage Reagents in commercial solutions are stored at 4°C. B6.2.2.2 PEROXIDASE ❶ Diaminobenzidine tetrahydro-chloride  Reagents/Solutions • DAB • 30% hydrogen peroxide • Tris–HCl/NaCl buffer; pH 7.6  Precaution Reagent dangerous if inhaled  Working solution a. Dissolve: • DAB or 1 tablet 10 mg • Revelation buffer 10 ml b. Filter the solution. c. To the filtrate, add: • 3% hydrogen peroxide/H2O 10 µl  Storage At −20°C, in aliquots, without hydrogen peroxide

B7 STAINING/COATING B7.1

Light Microscopy

B7.1.1

Cresyl violet

 Reagents/Solutions • Cresyl violet • Distilled water  Precaution Filter the solution before use.

380

➫ Inhibition of peroxidase activities ➫ There are other chromogen substrates that give different color precipitates (i.e., 4-chloro1-naphthol or 3-amino-9-ethylcarbazole). ➫ C12H14N4·4HCl ➫ 110 vol ➫ See Appendix B3. ➫ Tablet form preferable ➫ Prepare extemporaneously in darkness ➫ Mw = 360.12 ➫ Tablet of DAB to be dissolved in water

➫ Commercial solutions (DAB and H2O2) can be stored at 4°C.

Appendix B  Working solution Dissolve • Cresyl violet • Distilled water  Storage B7.1.2

➫ For some weeks

Eosin

 Reagents/Solutions • Distilled water • Eosin  Precaution None  Working solution Dissolve • Eosin • Distilled water  Storage

B7.1.3

1g 100 ml At rt

5g 100 ml At rt

➫ In an opaque container ➫ For several months

Harris’s hematoxylin

 Reagents/Solutions • Acetic acid • Alum • Distilled water • Ethyl alcohol • Harris’s hematoxylin • Yellow mercury oxide  Precautions Mercury oxide is toxic. Filter the solution before use. The solution matures with time.  Working solution a. Dissolve: • Hematoxylin 5g • Ethyl alcohol 50 ml b. Dissolve while warm: • Alum 100 g • Distilled water 1l c. Mix a and b d. Heat to boiling point, and add: • Mercury oxide 2.5 g e. Withdraw rapidly from the heat, and add: • Acetic acid 20 ml  Storage At rt

➫ C2H4O2 ➫ Aluminum potassium sulfate (AlK(SO4)2· 12H2O) ➫ C16H14O6

➫ This increases in its dyeing properties. ➫ Mw = 302.30 ➫ Mw = 474.39

➫ Mw = 60.05 ➫ For some months ➫ In an opaque container

381

Reagents B7.1.4

Methylene green

 Reagents/Solutions • Distilled water • Methyl green  Precaution Filter before use.  Working solution Dissolve: • Methyl green • Distilled water  Storage B7.1.5

➫ C27H35N3BrCl·ZnCl2

1g 100 ml At rt

Rapid nuclear red

 Reagents/Solutions • Aluminum sulfate • Distilled water • Rapid nuclear red  Precaution Filter the solution before use.  Working solution a. Prepare 1 l of aluminum sulfate at 5%: • Aluminum sulfate 50 g • Distilled water 1l b. Heat to boiling point. c. Withdraw from the heat, and add: • Nuclear red 1g  Storage At 4°°C B7.1.6

Toluidine blue

 Reagents/Solutions • Distilled water • Sodium tetraborate • Toluidine blue  Precaution Filter the solution before use.  Stock solution a. Mix and dissolve at room temperature: • Toluidine blue 1g • Sodium tetraborate 1g • Distilled water 100 ml b. Filter.  Working solution Dilute: • Stock solution 1 vol • Distilled water 50 vol  Storage At rt

382

➫ Mw = 653.2 ➫ For some weeks ➫ Not to be confused with Fast-Red ➫ Al2(SO4)3 ➫ C14H9NO7S

➫ Mw = 342.1

➫ Mw = 335.3 ➫ For some weeks ➫ 1% aqueous toluidine blue

➫ Na2B4O7·12H2O ➫ C15H16N3⋅SCl

➫ Mw = 305.80 ➫ Mw = 381.37

➫ For some months ➫ Filter before use

Appendix B

B7.2

Electron Microscopy

B7.2.1

Uranyl acetate

 Products • Ammonia • 95% ethanol • Oxalic acid • Sterile distilled water • Uranyl acetate  Precautions Uranium is radioactive. Avoid all contact with the skin. Avoid inhaling uranyl powder.

➫ Electron-microscopy quality (EM) ➫ NH4OH (10% solution) ➫ ➫ ➫ ➫ ➫

C2O4H2·2H2O See Appendix B1.1. (CH3COO)2 UO2·2H2O Use a fume hood. Emits α radiations

➫ Wear a mask while weighing out the uranyl acetate.

B7.2.1.1 2.5% ALCOHOLIC URANYL ACETATE  Protocol a. Prepare just before use in a brown glass container: • 5% aqueous uranyl acetate 1 vol • 95% alcohol 1 vol b. Filter in the dark. c. Stain in the dark.

➫ In a dark room with a safe light ➫ Saturated solution (see Appendix B7.2.1.2) ➫ Alcoholic uranyl is very sensitive to the light.

 Storage Do not store. B7.2.1.2 2 TO 5% AQUEOUS URANYL ACETATE  Protocol • Uranyl acetate 0.5–1.25 g • Distilled water Mix. • Filter using a Millipore.  Storage B7.2.1.3

➫ Dissolve at room temperature in the dark with a magnetic stirrer for 1 to 2 h.

25 ml 0.22 µm At 4°°C

➫ For 1 month away from light ➫ 4% aqueous uranyl acetate/300 mM oxalic acid; pH 7.5 ➫ Electron microscopy quality ➫ See Appendix B7.2.1.2.

4% NEUTRAL URANYL ACETATE

 Solutions • 4% aqueous uranyl acetate • 10% ammonia • 300 mM oxalic acid  Precaution Away from the light  Protocol a. Mix: • 4% aqueous uranyl acetate • 300 mM oxalic acid

➫ Stock solution

1 vol 1 vol 383

Reagents b. Adjust the pH • 10% ammonia c. Filter using a Millipore.  Storage B7.2.2

7–7.5 0.22 µm At 4°°C

➫ ➫ ➫ ➫

0.882 g

➫ Mw = 294.10 ➫ 1% sodium tartrate can also be used. ➫ Mw = 331.23 ➫ A homogenous precipitate forms. ➫ Mw = 40.00 ➫ The precipitate dissolves at a basic pH ≈ 10.

➫ For 1 month ➫ Fill the aliquots to the top to avoid contact with carbon dioxide in the air.

Methylcellulose

B7.2.3.1 2% METHYLCELLULOSE  Products • Methylcellulose (Tylose)  Solution • Distilled water  Precaution Do not stir. Take the quantity needed from the top of the tube.  Protocol • Methylcellulose 2g • Distilled water 100 ml

384

Electron microscopy quality Pb(NO3)2 C6H5Na3O7·2H2O NaOH

➫ See Appendix B2.20. ➫ See Appendix B1.1.

• Lead nitrate 0.662 g a. Dissolve the two products separately in distilled water. b. Mix the two solutions thoroughly. c. Add: • 1 N sodium hydroxide 4 ml d. Mix until the precipitate dissolves completely. e. Add: • Distilled water to 25 ml f. Filter with a 0.22 µm Millipore filter.  Storage In aliquots At 4°°C

B7.2.3

➫ For 1 month ➫ According to Reynolds

Lead citrate

 Products • Lead nitrate • Sodium citrate • Sodium hydroxide  Solutions • 1 N sodium hydroxide • Distilled water  Protocol • Sodium citrate

➫ Use pH paper; uranyl salts cause deterioration of the elecrodes.

➫ Stock solution ➫ Electron microscopy quality ➫ Tylose is the commercial name of methyl cellulose. ➫ See Appendix B1.1.

Appendix B a. Heat. b. Place with stirring. c. Place without stirring until the Tylose has dissolved. d. Centrifuge.  Storage

95°°C 15 min 4°°C overnight 15–30 days 4°°C 100,000 g 60 min At 4°°C At −20°°C

➫ The solution forms very slowly (>1 month). ➫ Not indispensable ➫ ➫ ➫ ➫

For several weeks Solution in use For several months Reserve solution

➫ A staining solution can also be used for coating sections of frozen tissue.

B7.2.3.2 0.8% METHYLCELLULOSE, 0.2% NEUTRAL URANYL ACETATE

 Solutions • Distilled water • 2% neutral uranyl acetate • Methylcellulose 2%  Precaution None  Protocol a. Add the methylcellulose and the neutral uranyl acetate to give a final concentration of 0.2%: • Neutral uranyl acetate 200 µl • Tylose 800 µl • Distilled water 100 ml b. Stir. c. Centrifuge.  Storage At 4°°C At −20°°C B7.2.4

➫ Make up stocks and keep at 4°C.

➫ See Appendix B1.1. ➫ See Appendix B7.2.1.3. ➫ See Appendix B7.2.3.2.

➫ Or more, if necessary ➫ ➫ ➫ ➫

Eliminates bubbles For several weeks Solution for use For several months

Sodium silicotungstate (0.5%)

 Solutions • Distilled water • Sodium silicotungstate  Precaution Avoid all contact with the skin.  Protocol Dissolve in distilled water: • Sodium silicotungstate • Distilled water  Storage

➫ See Appendix B1.1. ➫ AR quality

0.5 g 100 µl At 4°°C

➫ For several weeks

385

Reagents

B8 B8.1

MOUNTING MEDIA Aqueous

Aqueous mounts are used where the reaction precipitate is soluble in alcohol: • Buffered glycerine • Commercial media • Moviol B8.1.1

➫ See Appendix B3.4.2 or B3.4.3.

➫ For some days

Moviol

 Reagents/Solutions • Distilled water • Glycerol anhydride • Moviol 4–88 • 100 mM Tris-HCl buffer; pH 8.5  Precaution None  Preparation a. Mix: • Moviol 4–88 • Glycerol b. Add: • Distilled water c. Mix and let stand. d. Add: • Buffer e. Incubate. f. Mix, centrifuge.  Storage In aliquots

386

➫ See Appendix B8.1.2.

Buffered glycerine

 Reagents/Solutions • Glycerol • 100 mM phosphate buffer, or PBS  Precaution None  Preparation • Glycerol 1 vol • Buffer 1 vol  Storage At 4°°C B8.1.2

➫ See Appendix B8.1.1.

➫ RP quality ➫ C3H8O3 ➫ See Appendix B3.7.1.

2.4 g 4.8 ml

➫ Mw = 92.10

6 ml 2–48 h 60°°C

12 ml overnight 60°°C 5,000 g 15 min At −20°°C

➫ For some days

Appendix B

B8.2

Permanent

Permanent mounting media are used after dehydration and passage through xylene (or a similar chemical).  Reagents • Commercial media  Precautions Toxic on contact or by inhalation Polymerization on drying  Storage At room temperature in tightly closed containers

➫ Aromatic solvents, or substitutes ➫ Xylene can be used to make the solution more fluid.

387

Glossary

Glossary

A Adenine

Alkaline phosphatase Antibody

• Monoclonal

• Polyclonal

• Primary • Secondary

Antigen

Antigenic determinant Antiserum Araldite Artifact ATPase Autoclave Automatic freeze-substitution system (AFS)

Autoradiography/radioautography

➫ A puric base that is found in nucleosides, nucleotides, coenzymes, and nucleic acids. It forms two hydrogen bonds with thymine and uracil in double-stranded nucleic acids. ➫ A phosphatase extracted from calf intestine (Mw = 80 kDa). Its optimum pH is alkaline. ➫ A glycoprotein produced in response to the presence of an antigen. It can combine with the antigen that induced its production. ➫ An antibody that is specific to an epitope produced by a cell culture resulting from the fusion of a tumoral cell and an antibodyproducing cell. ➫ All the immunoglobulins contained in serum from an animal immunized with a given antigen. Polyclonal antibodies recognize several epitopes (or antigenic determinants). ➫ An antibody that is specific to the antigen being sought in an immunocytological reaction. ➫ An antibody used for indirect immunological reactions, whether conjugated or not, and directed against the animal species in which the antibody IgGs were produced. ➫ Any substance that is foreign to a receptor organism. It is said to be immunogenic when it induces an immune response (which can bring about the production of antibodies). ➫ See Epitope. ➫ A serum containing an immunoglobulin that is specific to an injected protein. ➫ An epoxy resin made up of chains of aromatic glycerol polyarylether rings. ➫ Background noise, or nonspecific reactions. ➫ Adenosine triphosphatase (an enzyme that catalyzes ATP hydrolysis). ➫ A device that sterilizes objects by the use of pressurized steam. ➫ This apparatus automatically dehydrates, impregnates, and polymerizes biological samples embedded in hydrophilic resin at low temperature. ➫ A method for detecting hybrids containing isotopes.

391

Glossary • Activity

• Efficiency • Resolution

Avidin

➫ The number of disintegrations per second in a radioactive source. The unit is the Becquerel (Bq), which corresponds to one disintegration per second. The previous unit was −11 the curie (Ci). (1 Bq = 2.7 × 10 Ci; 1 Ci = 7 3.7 × 10 disintegrations/second). − ➫ The number of silver grains obtained by β radiation. ➫ The distance between the location of a silver grain and a radiation source. It is defined by the energy of the isotope and the autoradiographic system. ➫ A glycoprotein (Mw = 16,400) produced in the uterus of the chicken. It contains four subunits, each of which binds a molecule of biotin 15 –1 with a very high affinity (Ka = 10 M ).

B Background noise Base

• Puric

• Pyrimidic

Base pair

Bright-field microscope

392

➫ A nonspecific signal (artifact). ➫ Nucleic acids contain two groups of bases: purines and pyrimidines. ➫ A base is a flat, cyclical molecule composed of hydrogen, carbon, and nitrogen. The initials that identify the genetic code are A (adenine), G (guanine), C (cytosine), T (thymine), and U (uracil). ➫ The puric bases are guanine and adenine, whose chemical structure resembles that of the pyrimidines, combined with a pentagonal structure (the pyrimidic ring + the imidazol ring). ➫ The pyrimidic bases are cytosine and thymine (DNA) or uracil (RNA), whose chemical structure is hexagonal. ➫ A parameter whereby the length of a double strand of DNA can be determined. A base consists of a nucleotide, and a base pair is a combination of two nucleotides, one on each of the two complementary DNA strands. ➫ A microscope whose object is illuminated directly and intensely, giving an image on a light-colored background.

Glossary

C Carbon film Cellular clone

Chelator

Chemography

Chromogen Chromosome

Cloning vector

Coating

Codon

• Initiation

• Termination

Collodion

Counterstaining

Cryofixation

➫ Carbon vaporized onto grids covered with collodion or formvar. ➫ All the cells that are produced by the division of a given mother cell (and which are thus genetically identical). ➫ An agent that captures bivalent cations 2+ (e.g., Ca ), thus blocking the action of enzymes (e.g., EDTA). ➫ The appearance of background noise in a photographic emulsion due to the chemical effect of a biological preparation. ➫ A component that produces a colored substance through an enzymatic reaction. ➫ From the Greek khroma, meaning “color,” and soma, meaning “body.” It contains most or all of the cellular DNA, and thus the genetic information. ➫ A DNA molecule that is capable of replicating itself (replicon). It is used to transport a piece of foreign DNA (e.g., a gene) into a receptor cell. It can be a plasmid, a phage, etc. ➫ A step that consists of placing a layer of saccharose or methylcellulose on a tissue sample or a section. ➫ A sequence of three bases (or triplets) in mRNA. It codes for an amino acid, and can also indicate the beginning or the end of a transcription process. ➫ A codon that initiates protein synthesis. There is only one such codon, i.e., AUG, which also codes for methionine. ➫ Or “stop”: a codon that does not code for an amino acid, but acts as a signal for the termination of a protein synthesis operation (e.g., UAA, UAG, UGA). ➫ An organic film used as a membrane support on a grid. Celloidin, also known as collodion, is a purified form of nitrocellulose. ➫ Light-microscopic staining that is used to obtain a different contrast from that of the hybridization signal, such that the general tissue structure is defined. ➫ The transformation of water in cells from a liquid to a solid state, with the stopping of cell functioning and the hardening of the sample.

393

Glossary Cryopolymerization Cryoprotectant

Cryoprotection

Cryosection Cryosubstitution

Cryoultramicrotome Cryoultramicrotomy Cytosine

➫ The hardening of a resin by ultraviolet radiation at low temperature. ➫ A molecule that, when added to a sample in the aqueous phase, allows it to be frozen in an amorphous state. ➫ The incubation of samples in a solution that limits crystallization during the freezing process. ➫ A section of frozen tissue. ➫ The penetration, at low temperature, of samples fixed in solvents, then in solvent– resin mixtures of increasing resin concentration, and finally in pure resin. ➫ An apparatus for cutting semithin and ultrathin sections of frozen tissue. ➫ A technique for cutting semithin and ultrathin sections of frozen tissue. ➫ A pyrimidic base that is a component of nucleosides, nucleotides, and nucleic acids.

D Dehydration

Denaturation

Denhardt’s solution

Deoxyribonucleic acid (DNA)

• Complementary (cDNA) • Ligase

394

➫ The progressive replacement of cell water through successive baths of increasing concentrations of solvent (e.g., ethanol or acetone). ➫ The separation of the two strands of a DNA double helix, either by heat or by the action of chemicals (with a change of conformation of the nucleic acids). It can also be applied to certain types of RNA. ➫ A mixture of polymers that, when added to the hybridization medium, reduces the background noise. ➫ Nucleic acid that is found in the nucleus of the cell (and which, in all cellular organisms, carries genetic information). The DNA molecule is a polynucleotide composed essentially of deoxyribonucleotides linked by phosphodiester bonds. ➫ A DNA copy of an RNA molecule. Generally messenger RNA. ➫ An enzyme in which two fragments of DNA are combined via the formation of a new phosphodiester bond.

Glossary • Polymerase Deoxynucleoside Deoxynucleotide Deoxyribose Deproteinization

Dextran

Dissecting needle

DNA DNase Duplication

➫ An enzyme that synthesizes a DNA molecule from a DNA template. ➫ A molecule composed of a base and a deoxyribose. ➫ See Nucleotide. ➫ A ribose that has lost an oxygen atom at 2′. ➫ An enzymatic treatment that partly eliminates proteins, and in particular those associated with nucleic acids. ➫ A macromolecule, generally in the form of a sulfate (dextran sulfate), which, when added to a hybridization buffer, increases the effectiveness of the hybridization process by concentrating the probe. ➫ A tool with an extrafine point (for delicate work), which facilitates the positioning of semithin or ultrathin sections. ➫ See Deoxyribonucleic acid ➫ An enzyme that destroys DNA in a specific way. ➫ A process whereby a DNA molecule copies itself exactly, either in vivo or in vitro.

E Electron microscope

ELISA (enzyme-linked immunosorbent assay) test Embedding

Emission energy Endogen Enzyme

➫ The type of microscope that is used to observe ultrathin tissue sections. Sections are placed on a grid and bombarded by a beam of electrons, which are deflected by substances of high atomic weights, thus giving rise to contrasts. ➫ A technique used to detect and quantify specific antibodies or antigens. ➫ The different steps (dehydration, impregnation, polymerization) leading to obtaining a block from which ultrathin sections of a sample can be cut. ➫ Energy released by the emission of radiation, expressed in MeV (mega-electronvolts). ➫ A molecule that is present in a biological sample. ➫ A protein that catalyzes a specific chemical reaction. Its name generally suggests its function, with the suffix “-ase” added to the name of the molecule upon which it acts (e.g., ribonuclease cuts RNA molecules, and protease hydrolyzes proteins).

395

Glossary Epitope

Epon Epoxy Exogen Exon

➫ Also known as “antigenic determinant.” It is the part of an antigen that is recognized by an antibody (an epitope is a structure that can combine with a single antibody molecule). An antigen generally has several epitopes, each corresponding to a sequence of three to six contiguous or noncontiguous amino acids. ➫ An epoxy resin. ➫ See Resin. ➫ A contaminant (in laboratory equipment, etc). ➫ A transcribed or coding sequence in a gene.

F Fab

F(ab′′ )2

Fc

Fixation

Formamide

Formvar

Freezing Freezing temperature

396

➫ An antigen-binding fragment obtained by using an enzyme (papain) to cut an antibody. Enzymatic cutting takes place upstream from the disulfide cross-links that exist between the two heavy immunoglobulin chains (Mw = 45 kDa). ➫ A fragment obtained by using an enzyme (pepsin) to cut an antibody. It represents two antibody sites (Mw = 100 kDa). ➫ A fragment that has no antibody activity, but that crystallizes easily. It is specific to the species that was the source of the immunoglobulins (Mw = 50 kDa). ➫ Its purpose is to stabilize cell metabolism and deactivate endogenous RNase, while conserving cell morphology and the integrity of nucleic acids. ➫ A chemical that favors the denaturing of nucleic acids. The addition of 1% formamide to the hybridization solution lowers the hybridization temperature by 0.65°C for DNA and 0.72°C for RNA. ➫ A strong organic film that serves as a membrane support on a grid before ultrathin sections are placed on slides. ➫ The transition from the liquid state to the solid state. ➫ The temperature of a liquid–solid mixture. The temperature at which a sample freezes.

Glossary

G Gene

Genetic engineering

Genome Genotype Glutaraldehyde

Guanine

➫ The unit of genetic information that codes for a polypeptide or a molecule of rRNA or tRNA. The complete set of genes is known as the “genome.” ➫ The various techniques used to modify an organism’s genetic information by changing its genomic nucleic acids. ➫ The complete set of genes of a cell or a virus. ➫ The sum of the information contained in the genes of a given organism. ➫ A reticulating fixative that gives rise to cross-links between different cell and tissue structures. ➫ A puric base that is found in nucleosides, nucleotides, and nucleic acids.

H Half-life of a radioelement Hapten

Haptenization

Histones

Hybridization of nucleic acids

Hydrophilic

Hydrophobic

➫ The time required for the radioactivity of an isotope to decline by half. ➫ A molecule that, although non-immunogenic, can induce the production of antibodies directed against itself when coupled to a macromolecular carrier. ➫ The labeling of nuclear probes by haptens, which are introduced either by chemical reactions or enzymes. ➫ Proteins that are rich in arginine and lysine, associated with DNA in eukaryotic chromosomes. ➫ The formation of hybrid molecules: doublestranded DNA, DNA–RNA, or RNA–RNA. As long as the sequences are complementary, such hybrids are stable. ➫ Describes a polar substance that has a strong affinity for water, or that dissolves easily in water. ➫ Describes a nonpolar substance that has no affinity for water, or that does not dissolve easily in water.

397

Glossary

I Immunocytochemistry

Immunocytology

Immunoglobulin • G immunoglobulins (IgG)

• M immunoglobulins (IgM)

Impregnation

Intron

Isotope

➫ A science that provides cytological evidence of antigenic constituents by means of immunochemical reactions. ➫ A science that provides cytological evidence of antigenic constituents by means of immunocytological reactions. ➫ See Antibody. ➫ A class of immunoglobulins that are predominant in serum (>85% of the immunoglobulins in serum) (Mw = 150 kDa). ➫ A class of serous immunoglobulins (Mw = 900 kDa) made up of heavy chains and light chains such as IgGs. They take the shape of a five-pointed star with a central ring. ➫ Or “substitution.” The gradual replacement, in tissue, of one fluid by another (e.g., alcohol by resin). ➫ A noncoding intercalary sequence of an interrupted gene that is transcribed into heterogeneous nuclear RNA (hnRNA, or primary transcripts of DNA), but is cut out during the splicing that brings about the maturation of mRNA. ➫ One of two or more forms of a given element that have different atomic weights but similar chemical properties.

K Knifemaker

➫ An apparatus for producing glass knives of the kind that are used to make frozen and/or resin-embedded tissue sections.

L Lowicryls

• Hydrophilic — Lowicryl K4 M — Lowicryl K11 M 398

➫ These resins are composed of a mixture of acrylate and methacrylate monomers. Their advantage is that they remain highly fluid at low temperatures. Polymerization at –20°C, or –80°C, under ultraviolet radiation (360 nm). ➫ Or “polar.” These resins all have the same viscosity. ➫ Polymerization at –20 or –35°C. ➫ Polymerization at –60°C.

Glossary • Hydrophobic — Lowicryl HM 20 — Lowicryl HM 23 LR White (“London Resin Gold”)

➫ Or “nonpolar.” ➫ Polymerization at –40°C. ➫ Polymerization at –80°C. ➫ An acrylic resin composed of a mixture of methacrylate and a hardener, specially made to be highly hydrophilic. Polymerization may take place in two ways (at 4°C or 50°C), depending on the required degree of hardness.

M Melting temperature (Tm)

Mer Methacrylate

➫ The temperature at which 50% of doublestranded DNA separates out into single strands. ➫ A unit of nucleic acid that can be a nucleotide or a pair of nucleotides. ➫ A monomer of aromatic polyhydroxyl acrylic resin.

N Nanometer (nm) Nonspecificity

Northern blot technique Nucleic acid

• Complementary • Exogenous

• Genomic • Mitochondrial • Targets Nucleoside

➫ 1/1000 micrometer, or 10 meter. ➫ Mismatching: either nonhomogeneous (probe-noncomplementary nucleic acid) or heterogeneous (protein-nucleic acid). ➫ Detection by hybridization of specific RNA fragments transferred onto a membrane. ➫ A macromolecule that carries genetic information. There are two types of nucleic acid: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). ➫ Nucleic acids that can be used as probes. ➫ Nucleic acids that are present in a cell, but that do not belong to its genome or take part in its expression. Origin: viral, bacterial, or fungus. ➫ Nucleic acids that contain the genetic characteristics of a cell. ➫ Nucleic acids that are present in mitochondria. ➫ Nucleic acids that are sought in a cell. ➫ A molecule made up of a puric or pyrimidic base and a sugar (ribose or deoxyribose). –9

399

Glossary Nucleosome

Nucleotide

➫ A DNA molecule associated with basic proteins (histones) in the eukaryotic nuclear matrix. Histones participate in the formation of chromatin, which consists of a flexible chain of repeated units, namely, the nucleosomes. ➫ A nucleoside + one or more phosphate groups.

O Oligonucleotide

Osmium tetroxide (OsO4)

➫ From the Greek oligos, meaning “small.” Oligonucleotides are short fragments of DNA, with 15 to 60 nucleotides. The term is generally used for syntheses. ➫ A highly reductive chemical fixative (sometimes incorrectly called osmic acid).

P Palindrome

Peptide Phenotype

Phosphatase Phosphorylation PLT

Poly (A)

Polymerase • DNA polymerase • RNA polymerase • Thermostable Polymerase chain reaction (PCR)

400

➫ A complementary sequence contained in a given strand of DNA, forming an intrachain hybrid. ➫ A short chain of amino acids. ➫ A set of characteristics that can be identified by experimentation. The physical expression of a genotype. ➫ An enzyme that hydrolyzes phosphate groups in molecules. ➫ The addition of a phosphate group by the action of an enzyme. ➫ “Progressive lowering temperature,” according to the progressive low-temperature dehydration method, using liquid nitrogen vapor. ➫ A repetitive polynucleotide (A) sequence that is attached by the action of a ligase to the 3′ end of a transcribed mRNA molecule. This is a characteristic property of mRNA. ➫ A synthesization enzyme. ➫ For DNA. ➫ For RNA. ➫ Able to withstand high temperatures. ➫ The amplification of a given DNA sequence by repeated cycles of denaturation, hybridization, and elongation.

Glossary Polymerization

Polymerization chamber Polynucleotide Polynucleotide kinase

Postfixation Precipitation reaction

Prehybridization Pretreatment Primer

• Anti-sense • Sense

Probe

• Anti-sense

• Non-sense

• Oligonucleotide

• Sense

➫ A process during which resin changes from its original liquid state to a solid state under the influence of factors such as catalysts, heat, and ultraviolet radiation. ➫ An apparatus that uses ultraviolet radiation to harden resin. ➫ A chain made up of several nucleotides. RNA is a polynucleotide. ➫ The kinase enzyme. It is extracted from calf thymus, and is used in the radioactive labeling of oligonucleotide probes by phosphorylating the 5′ end, with a phosphate group in the γ position. ➫ A complementary fixation step. ➫ A reaction that brings about the insolubilization of a nucleic acid using alcohol and salt. ➫ The incubation of sections in a hybridization solution without a probe. ➫ A step that is carried out before hybridization. ➫ An oligonucleotide sequence that acts as the starting point for the neosynthesis of nucleic acids through the action of DNA polymerase. ➫ A complementary sequence at the 3′ end of the sequence being studied. ➫ A sequence that is complementary to the 5′ end of a sequence that is complementary to the sequence being studied, or a sequence at the 5′ end of the sequence being studied. ➫ A fragment of nucleic acid (DNA or RNA) whose nucleotide sequence is complementary to that of the nucleic acid being sought, which is immobilized in the preparation (target). ➫ A sequence that is complementary to that of the target with which it specifically hybridizes. ➫ A sequence that is complementary to, but of the same sense as, the target. It serves as a negative control. ➫ If a sequence is known, it is possible to make a single-strand probe, using synthetic oligonucleotides (≈30 nucleotides), whose sequence is complementary to that of the nucleic acid being sought (the target). ➫ A homologous copy of the target. It serves as a negative control. 401

Glossary Promoter

Protein Proteinase K Purine

Pyrimidine

➫ A sequence in a sense strand of DNA, situated upstream from a gene to which polymerase RNA links before the start of the transcription process. ➫ A macromolecule composed of amino acids. ➫ An enzyme that hydrolyzes proteins to form amino acids. ➫ A basic nitrogenous molecule comprising two aromatic nuclei essentially made up of the bases adenine (A) and guanine (G). It is found in nucleic acids and other cell components. ➫ A basic nitrogenous molecule with an aromatic nucleus, made up essentially of the bases cytosine (C), uracil (U), and thymine (T). It is found in nucleic acids and other cell components.

R Radioactivity Radioautography Recombinant DNA technology

Renaturation Replication

Resin

• Acrylic

• Epoxy

• Hydrophilic

402

➫ The emission of radiation by certain elements, which thereby turn into other elements. ➫ See Autoradiography. ➫ A set of techniques used in genetic engineering for the identification and isolation of a specific gene, its insertion into a vector such as a plasmid to form recombinant DNA, and, finally, the production of large quantities of the gene and its product. ➫ The rematching of a complementary nucleic acid. ➫ A process during which an exact copy of a DNA or RNA molecule is synthesized from a DNA or RNA template. ➫ A monomer of the epoxy and/or acrylic type, used for embedding tissue to make ultrathin sections. ➫ Lowicryls, LR White, Unicryl, etc. These resins are composed of acrylates or methacrylates, which are highly fluid until polymerized. They have excellent penetrative properties. ➫ Epon, Araldite, Spurr, etc. These resins are composed of hydrophobic monomers whose polymerization results in very hard blocks. ➫ For example, acrylic resins. This type of resin polymerizes in the presence of a small amount of water.

Glossary • Hydrophobic

• Viscosity

Ribonuclease Ribonucleic acid (RNA)

• Complementary RNA (cRNA) • Messenger RNA (mRNA)

• Polymerase

• Pre-messenger • Ribosomal RNA (rRNA) • Splicing

• Transfer RNA (tRNA) Ribose Ribosome

RNase

RNase-free conditions

➫ For example, epoxy resins. This type of resin does not polymerize in the presence of water. ➫ The viscosity of acrylic resins is low (e.g., methacrylate: 0.7 centipoises at 25°C), whereas that of epoxy resins is high (e.g., Araldite: 1300 to 1650 centipoises at 25°C). ➫ An enzyme that breaks down RNA; see RNase. ➫ A molecule that carries genetic information. It is very similar to DNA. The sugar molecule in RNA is a ribose rather than, as in the case of DNA, a deoxyribose. A polynucleotide composed of ribonucleotides joined by phosphodiester bonds. It can take different forms: messenger RNA, transfer RNA, ribosomal RNA, or viral RNA. ➫ An RNA copy of an RNA molecule, generally obtained by in vitro transcription. ➫ Single-stranded RNA, which is synthesized from a DNA template during the transcription process. It binds to ribosomes and carries the messages required for protein synthesis. ➫ An enzyme that catalyzes the synthesis of RNA from a DNA matrix. RNA polymerase recognizes errors resulting from mismatching. Its exonucleasic activity allows it to replace incorrect nucleotides with the correct ones. ➫ RNA that is present in the nucleus and the cytoplasm. ➫ The basic component of ribosomes, which are directly involved in protein synthesis. ➫ A nuclear process during which introns are cut out of primary mRNA transcripts during its formation. ➫ A short RNA chain that transports amino acids during protein synthesis. ➫ A ribonucleic acid. In RNA, a sugar. ➫ An organelle in which proteins are synthesized and in which the messages coded in mRNA are translated. ➫ See also Ribonuclease. An enzyme that breaks down single-stranded RNA only. RNase treatment carried out after hybridization reduces background noise, and serves as a control. ➫ Experimental conditions in which all contamination by exogenous ribonuclease is eliminated, to preserve mRNA. 403

Glossary

S Saline concentration Salinity

Sections • Semithin

• Ultrathin Sensitivity

Sequence being sought Serum • Immune • Nonimmune • Preimmune Signal Southern blot technique

Specific activity

Specificity Spliceosome Stability

Stain for electron microscopy • Negative

404

➫ Ionic strength. See Salinity. + ➫ The concentration of Na ions, which affects the stability of hybrids. The hybridization speed increases with the concentration of salts. ➫ See Ultramicrotomy. ➫ 0.5- to 2-µm-thick sections placed on glass slides and observed at the tissue and cell levels by light microscopy. ➫ 60- to 100-nm-thick sections placed on a grid for electron microscopy. ➫ This represents the smallest quantity of target nucleic acid that can be detected in a cell or tissue, or the number of molecules that can be detected by a given label. ➫ A sequence with a primer at each end. ➫ Defibrillated plasma. ➫ Serum from an animal after immunization. ➫ Serum from a nonimmunized animal of a given species. ➫ Serum from an animal before immunization. ➫ An in situ PCR/RT-PCR reaction product that shows the location of an amplified product. ➫ Detection by hybridization using a labeled probe of specific DNA fragments transferred onto a membrane. ➫ The specific activity of a probe results from its labeling, i.e., the number of isotopes or antigens incorporated, by comparison with the mass or the concentration of the probe. ➫ Total complementarity in matching between two nucleic acids. ➫ A site where the splicing of an mRNA precursor takes place. ➫ Relationship between two molecules of nucleic acid, depending on their nature. The three types of duplex that can be formed, in increasing order of stability, are DNA–DNA, DNA–RNA, RNA–RNA. ➫ Salts of heavy metals (e.g., uranium or lead), or tungstic acid. ➫ Heavy metal salts, which are deposited in the spaces between structures, and which thus appear light-colored against a black background.

Glossary • Positive

Sterilization

Streptavidin

Stringency

Substrate

➫ Heavy metal salts, which are deposited onto ultrathin sections so that the structures can be observed before carrying out electron microscopy. The structures appear black against a light-colored background. ➫ A process whereby an organism, a microorganism, or an enzyme is either destroyed or eliminated from an object or a solution. ➫ A protein of bacterial origin that has a very weak charge and a high affinity with four biotin molecules, and that generates little background noise. Its characteristics are similar to those of avidin, except that it has a neutral isoelectric point, and no affinity with lectins. ➫ A parameter that is used to express the efficiency of hybridization and washing conditions (depending on the concentrations of salt and formamide, and the temperature). A low level of stringency favors nonspecific matchings, whereas too high a level gives rise to a specific signal of lower intensity. ➫ A substance on which an enzyme acts.

T Target Terminal deoxynucleotidyl transferase (TdT)

Thymine Transcriptase

Transcription Transgenic

Translation Triphosphate nucleotide

➫ The nucleic sequence being sought within a cell. ➫ An enzyme that is used for labeling oligonucleotide probes by elongation of the 3′ end, provided that this is hydroxylated (free –OH). It can polymerize NTP and dNTP. ➫ A pyrimidic base that is found in nucleosides, nucleotides, and DNA. ➫ An enzyme that catalyzes transcription. In RNA viruses it is an RNA-dependent RNA polymerase, which is used to make mRNA copies based on RNA genomes. ➫ A process in which single-stranded RNA is synthesized from a DNA template. ➫ Describes an animal or plant whose genome contains new genetic information in stable form, due to the acquisition of foreign DNA. ➫ The reading of the genetic code during protein synthesis. ➫ A nucleoside + three phosphate groups (e.g., ATP, GTP, CTP, TTP, or UTP).

405

Glossary

U Ultramicrotome

Ultramicrotomy Unicryl

Uracil Uridine

➫ An apparatus for cutting resin-embedded tissue sections of variable thickness: semithin (0.5 to 2 µm) or ultrathin (80 to 100 nm). ➫ A method for producing ultrathin embedded tissue sections, using an ultramicrotome. ➫ A highly hydrophilic methacrylate resin of low viscosity, giving rapid penetration of tissue. It polymerizes either at low temperature under ultraviolet radiation (–25 to –35°C) or with the application of heat. ➫ A pyrimidic base that is found in nucleosides, nucleotides, and RNA. ➫ A nucleotide composed of uracil.

V Viscosity

406

➫ The resistance of a fluid to flowing, or, in the case of a resin, to penetrating tissue. It is generally expressed in poises.

Index

Index

A Acetate ammonium, 136, 356 sodium, 136, 362 uranyl, 231, 383 Acetylation, 59 Acrylic resin, 188, 190, 225, 375 Adenine, 123 AEC, 164 Agarose, 353 gel, 101, 137, 353 Alkaline phosphatase, 154 NBT/BCIP, 161, 162 endogenous, 60, 155, 162, 163, 378 revelation, 161 3-Amino-9-ethylcarbazole, see also AEC Amplification of genomic DNA, 266 Amplified product, 11 diffusion, 11, 268 AMV, 74 Antibodies, see also Immunoglobulin Antibody conjugation, 155 Anti-sense, 6, 72, 98, 125 Araldite, 188, 224 Artifact autoradiography, 117, 167 Autoclave, 351 Autoradiography, 165 artifact, 11 developer, 377 development, 166 efficiency, 165 emulsion characteristics, 166 fixative, 377 macro-autoradiography, 168 film, 169 principle, 168 protocol, 169 material emulsion, 166 film, 169 micro-autoradiography, 168 emulsion, 171 principle, 171 protocol, 172 principle, 165 protocol macroscopy, 169 light microscopy, 172 quantification, 167 resolution, 128, 167

B Background, 237 BCIP, 162 Biotin, 96 affinity, 96 conjugation, 97, 155

endogenous, 96 visualization, 153, 155 Blocking agents, 222, 378 principle, 155, 159 protocol, 160 Blocking solution, 160, 221, 378 5-Bromo-4-chloro-3-indolyl phosphate, see also BCIP Buffer, acetylation, 364 blocking, 160, 221 cacodylate, 364 DNase I, 365 hybridization, 140 light microscopy, 142, post-embedding method, 228 pre-embedding method, 218 phosphate, 365 SSC, 367 Tris-EDTA, 367 Tris-HCl, 368 Buffered glycerin, 56

C cDNA probe, 126, 138 RT, 69 Cells, 40 culture, 40, 42 fixation protocol, 40, 43, 45 fixation, 43 protocol monolayers, 43 pellets, 40, 45 smears, 44 suspension, 40 pellet, 40, 45 smear, 40 Chloride calcium, 356 cobalt, 133 lithium, 136, 360 magnesium, 80, 106, 360 manganese, 108 sodium, 142, 144, 363 5-Chloro-2-methoxy-benzene-diazonium chloride, see also Fast Red Chromogens, 379 alkaline phosphatase, 379 peroxidase, 380 CoCl2, see also cobalt chloride Cofactor MgCl2, 80, 106 MnCl2, 108 CoCl2, 133 Collodion film, 226 Colloidal gold, 155, 229 Concentration Na+, 140 Contamination, 268

409

Index Controls, 256 detection, 260 hybridization, 259 other techniques, 261 PCR, 259 pretreatments, 257 probe labeling, 126 reagents, 256 results, 260 reverse transcription, 258 tissue, 257 tools, 256 Counterstaining electron microscopy, 231 protocols, 231 light microscopy, 174 protocols, 174 Cover disk/cover clip protocol, 82 system, 79 cRNA, see also RNA probe Cryo-embedding, 33 Cryogenic agents, 32, 202 principles, 32 type, 32, 202 Cryo-infiltration, 226 Cryopolymerization, 226 Cryoprotection, 32, 192, 200 agents, 32, 200 protocol, 32, 201, 202 Cryoprotective agents, 32 principles, 32 type, 32, 200 Cryoultramicrotomy, 192 Culture, 40 cell suspension, 40, 197 coverslide, 40 flask, 42, 198 slide, 42 Cytosine, 123

frozen tissue, 57 pepsin, 56, 57 pre-embedding method, 205 principle electron microscopy, 205 light microscopy, 54 problems, 272 pronase, 56, 57 proteinase K, 128, 163 vibratome sections principle, 205 protocol, 206 Detection, 147 autoradiographic, 165 biotin, 159 immunohistolochemical, 152 direct, 156 indirect, 156 problems, 275 Developer, 166, 169, 172, 377 Dewaxing, 53 3′-Diaminobenzidine tetrachloride, see also DAB Diethylpyrocarbonate, see also DEPC Digestion of DNA, 61 Digoxigenin characteristics, 97 nucleotide conjugated, 98 Dithiothreitol, see also DTT DMSO, 33, 111 DNA, 359 double-stranded, 5, 8, 125, 130 polymerase Extapol®, 108 inhibition, 107 Pfu®, 107 Taq®, 105 Tgo®, 107 Tth®, 108 repair, 264 single-stranded, 5, 9, 10, 126, 133 DNase, 61, 206, 359 dNTP, 73, 96 DTT, 80, 141, 358

D DAB formula, 163 principle, 123 protocol, 164 Dehydration, 62 Deionized formamide, 141, 217, 228, 357 Denaturation chemical, 228, 229 principles, 216, 229 probe, 137, 143 protocol, 137, 143 Denhardt’s solution, 141, 217, 228 DEPC, 141, 217, 228, 355 Deproteinization, 54 chemical treatment, 58

410

E Easyseal, 79, 83, 104 protocol, 83 system, 79 EDTA, 359 Efficiency autoradiographic, 167 label, 135 Electron microscopy, 177 applications, 190 choice of the method, 193 methods non-embedding, 191 post-embedding, 188

Index pre-embedding, 185 principle, 183 Electrophoresis, 101, 353 Embedding acrylic resin, 226 AFS, 226 cryo-embedding, 33 cryo-infiltration, 226 epoxy resin, 224 floating sections acrylic resin, 226 epoxy resin, 224 Lowicryl, 226 LR White, 225, 226 paraffin, 37 protocols acrylic resin, 226 epoxy resin, 224 vibratome sections acrylic resin, 226 epoxy resin, 224 Emission energy, 128, 129 Emulsion exposure, 166, 170, 174 Emulsion photographic characteristics, 171 coating, 173 detection, 174 dilution, 173 drying, 173 exposure, 166 melting, 173 storage, 173 thickness, 172 Enzyme alkaline phosphatase, 60, 154 endogenous, 60, 155, 162, 378 inhibition, 60 DNA polymerase Extapol®, 108 inhibition, 107 Pfu®, 107 Taq®, 105 Tgo®, 107 Tth®, 108 DNase, 61, 206, 359 peroxidase, 154 endogenous, 60, 222 inhibition, 60 Reverse transcriptase, 74 AMV, 74 M-MLV, 75 Tth®, 75 RNase, 362 Tth® DNA polymerase, 75 Epi-illumination, see also Epipolarization Epipolarization, 175 Epon, 224 Ethylene diamine tetraacetic, see also EDTA Exposure time, 166, 170, 174 Extapol® DNA polymerase, 108

3′ Extension, 132 different stages, 133 principle, 133 protocol antigenic probe, 135 radioactive probe, 134

F F(ab′)2, 153 Fab, 153 False negative, 269 False positive, 263 Fast-Red, 162 formula, 162 FITC, see also Fluorescein Fixation, 26 criteria for choosing, 26 freezing, 31 immersion, 31 parameters, 26, 27 perfusion, 31 post-fixation, 59, 207, 216 principles, 31 problems, 271 protocol, 31 cell culture, 198 cell suspension, 197 perfusion, 31 tissue, 31 whole animal, 31 Fixatives, 27, 371 chemical, 27, 221 criteria for choosing, 30 cross-linker, 27 electron microscopy, 196 formaldehyde, 28, 371 formol, 371 glutaraldehyde, 29, 196, 371 mixture, 30 osmium tetroxide, 221, 223 paraformaldehyde, 28, 372 precipitative, 27, 30 protocol, see also Fixation types, 27 Floating section, see also Vibratome section Fluorescein characteristics, 98 labeling control, 137 Formamide deionized, 141, 217, 228, 357 dimethyl, 161 Formvar film, 226 Freezing, 31 apparatus, 33 cryogenic agents, 32 principles, 31 protocol, 32

411

Index rapid, 33 techniques, 33 Frozen sections, 32 Frozen tissue technique, 32

G Glutaraldehyde, 29, 196 Glycerol, 33 Guanine, 123

H Half-life, 128, 129 Hot start, 114, 214 Hybridization, buffer, 141 cDNA, 126, 138 constituants, 141 cRNA, 140 electron microscopy, 216 frozen tissue technique, 140 oligonucleotide, 140 parameters, 138, 143 post-embedding technique, 218, 227 post-treatments, 143 pre-embedding technique, 216 principle, 216, 227 protocol, 228 principles, 123 probes, 125 protocol, 140 temperature, 138 tools, 125 Hybrids antigenic, 145 biotinylated, 145 matched, 143 mismatched, 143 radioactive, 145

I IgG, see also Immunoglobulin Immunocytology, principle, 229 protocol, 230 Immunoglobulin, 153 advantages/disadvantages, 157 anti-biotin, 154 principles, 156 protocol, 158 Immunohistochemistry, 152, 220 biotin, 159 IgG, 153 indirect principles, 156 protocol, 158,

412

pre-embedding method, 220 direct reaction, 222 indirect reaction, 223 reaction direct principles, 156 protocol, 158 streptavidin, 153 tool, 152 In vitro transcription, 127 Incorporated radioactivity, 137 Infiltration, 224, 226 Ionic strength, 140, 143

K K4M, see also Lowicryl

L Labeling the probe, 127 PCR, 129 antigenic label, 135 3′ extension, 132 principle, 129, 132 protocol, 130 radioactive label, 134 purification, 135 storage, 137 Labels, see also Biotin, Digoxigenin, Fluorescein, 35S, and 33P advantages/disadvantages, 127 antigenic, 97, 127 advantages/disadvantages, 127 characteristics, 97 controls antigenic probe, 137 radioactive probe, 137 criteria for choosing efficiency, 165 resolution, 103, 128, 167 sensitivity, 165 emission energy, 128, 129 enzymatic alkaline phosphatase, 154 peroxidase, 154 principles of visualization, 161, 163 fluorescent, 155 control, 137 FITC, 98 linkage, 103 particle, see also Colloidal gold position on nucleotide α, 134 γ, 102 of label, antigenic, 97

Index 33

P, 103, 134 S, 103, 134 radioactive, see also 35S, 33P advantages/disadvantages, 128 controls, 137 emission energy, 128, 129 half-life, 128, 129 position, 128 primers, 102 storage, 137 Latensification of colloidal gold, 155 Lead citrate, 231 Liquid nitrogen, 32 London resin gold, see also LR White Lowicryl, 226 LR White, 226 embedding, 226 35

M Melting temperature, see also Tm Methyl green, 174 protocol, 174 Methylcellulose, 193, 384 MgCl2, 80, 106, 360 M-MLV, 75 MnCl2, 108 Monolayer, 43 Mounting medium, 386 aqueous, 162, 163, 165, 386 permanent, 164, 175, 387

N Na thiosulfate, 174 NaOH, 228, 229, 363 NBT-BCIP, 161 protocol, 162 Nitroblue tetrazolium, see also NBT Nonspecific hybridization, 266 label incorporation, 264 synthesis, 265 Nucleic acids, see also DNA, RNA Nucleotides, 73, 96 antigenic, 97, 98 radioactive, 99, 103, 134

O Observations, see also Chapter 11 bright field, 175 dark field, 175 epipolarization, 175 principles, 175 Oligonucleotide, see also Probes characteristics, 125

construction, 126 criteria for choosing, 126 determination, length, 126 percentage G-C, 126 purification, 126 specific activity, 137 Osmium tetroxide, 221

P 33

P, 99, 129 advantages/disadvantages, 103, 129 characteristics, 129 Paraformaldehyde, 29 depolymerization, 29 PCR, 87 cycle first, 116, 215 last, 116, 215 number, 115, 215 phases, 115, 215 electron microscopy, 211 principle, 211 protocol, 219 enzymes, 104 characteristics, 107 criteria of choice, 109 Extapol®, 108 inhibition, 107 Pfu®, 107 Taq®, 105 Tgo®, 107 Tth®, 108 hot start, 114, 214 principle, 91, 94 problems, 273 protocol cell suspension, 117 tissue, 112 reaction mixture direct PCR, 112 indirect PCR, 113 RT-PCR, 93 types asymmetric, 9 in situ reaction direct, 13 avantages/disadvantages, 12, 15 protocol, 112 indirect, 15 protocol, 113 nested, 9 quantitative, 9 semiquantitative, 9 symmetric, 8 Pepsin, 56, 361 Permeabilization, 53 agents, 54, 205

413

Index principles, 53, 204 protocol, 54, 205 Peroxidase, 154, 163 endogenous, 60, 222, 379 inhibition, 60, 379 Pfu® DNA polymerase,107 Photographic support, see also Emulsion or Autoradiography Poly (A), 360 Polymerase chain reaction, see also PCR Polymerization cryopolymerization, 226 epoxy resin, 225 LR White, 226 Poly (T), 10, 70 Post-embedding technique, 188 Precipitation, 135 ammonium acetate, 136, 356 ethanol, 136 sodium acetate, 136 Pre-embedding technique, 185 Preparation, 23 cells, 40 culture, 42 monolayer, 42 suspension, 45 tissue, 25 Pretreatment, see also Fixation acetylation, 59 consequence, 63 dehydration, 62 denaturation, 137, 229 deproteinisation, 54 dewaxing, 53 permeabilization, 53 post-fixation, 59, 207 pre-embedding technique, deproteinization, 205 permeabilisation, 202 principles, 185 principle, 51 slides, 352 Primers, 6, 99 anti-sense, 6, 98 labeled, 102 5′ extension, 103 antigenic, 104 radioactive, 102 poly (T), 10, 70 random, 10, 71 sense, 6, 98 specific, 10, 72 characteristics, 72, 100 concentration, 101 hybridization temperature, 73, 100 position, 73, 100 storage, 101 validation, 101 Probes, 125 anti-sense, 125

414

characteristics, 125 controls, 126 definition, 125 denaturation, 137 DNA double-stranded, 126 single-stranded, 126 labeling techniques, 127 length, 126 oligonucleotide characteristics, 125 construction, 126 criteria for choosing, 126 length, 126 percentage G-C, 126 purification, 126 specific activity, 137 post-treatment, 143 RNA, see also In vitro transcription sense, 125 storage, 137 type, 126 utilization, 137 washes, 143 Problems, see Chapter 9 Pronase, 56, 361 Proteinase, 360 Proteinase K, 55, 360 concentration, 55 parameters, 55 protocol, 56 use, 55 Purification, 135 protocol, 136

R Resin acrylic, 225 Lowicryl, 225 LR White, 225 Resolution autoradiography, 167 Revelation, 147, 377 autoradiography, 165 immunohistochemistry, 152 principle, 150 Reverse transcriptase, 74 AMV, 74 criteria of choice, 76 M-MLV, 75 Tth®, 75 Reverse Transcription, 9, 65 electron microscopy, 207 pre-embedding method, 207 principle, 208 protocol, 209 principle, 69 problems, 272

Index protocol, 80 cell suspension, 81, 85 tissue section, 81, 86 tools, 70 primer, 70 dNTP, 73 enzyme, 74 materials, 77 types asymmetrical, 9 differential display, 9 symmetrical, 9 RNA, 361 RNase A, 362 RNase free conditions, 349 RNasin, 80 RT, see also Reverse transcription

S 35

S, 128 advantages/disadvantages, 128 characteristics, 128 Safe-light, 172 Sample electron microscopy, 195 cell, 195 tissue, 196 light microscopy, 21 cell, 40 tissue, 25 preparation, see also Fixation Sarcosyl, 362 Sealing system, 79 cover disk/cover clip, 79 easyseal, 79 protocol, 110 cover disk/cover clip, 82 easyseal, 83 Sections frozen, 34 paraffin, 38 staining, electron microscopy, 231 light microscopy, 174 storage thin, 62 ultrathin, 232 vibratome, 202 ultrathin, 226 vibratome, 199 parameters, 199 storage, 202 Sense, 6, 218 Sensitivity, 243 Sequence, 5 anti-sense, 6 sense, 6 target, 5, 11

Signal/background ratio, 237 Slides preparation, 352 Sodium hydroxide, see also NaOH Specific activity, 137 Specificity, 243 SSC, 367 Stabilization of structures, see also Fixation Staining electron microscopy, 231, 383 lead citrate, 231, 384 uranyl acetate, 231, 383 light microscopy, 380 cresyl violet, 380 eosin, 381 hematoxylin, 381 methyl green, 174, 382 nuclear red, 382 toluidine blue, 174, 382 Sterilization, 351 Storage primer, 111 probe, 137 sample, 34 solutions, see Appendices thin section, 62 ultrathin section, 232 vibratome section, 202 Streptavidin, 153, 155 Streptavidin/biotin complex, 153, 155 Sucrose, 32

T Taq® DNA polymerase, 105 Target sequence definition, 5, 11 destruction, 270 TdT, see also Terminal deoxytransferase Techniques for freezing tissue, 32 Techniques for labeling, see also 5′ Extension, 3′ Extension, and Symmetric PCR Terminal deoxinucleotide transferase, 132 Tgo® DNA polymerase, 107 Thermocycler materials, 77, 78, 109 programming, 116 Thymidine, 123 Tm, 138 Toluidine blue, 174 Tris, 364 Triton X-100, 364 Tw, see also Wash temperature Typical protocols, see Chapter 10

U Ultramicrotome, 226 Ultrathin section, 226, 231, 232 Uracile, 123

415

Index

V Vibratome equipment, 199 section embedding epoxy resin, 224 LR White, 226 parameters, 199 protocol, 201 storage, 202 Visualization alkaline phosphatase, 161 autoradiography, 165 macro-autoradiography, 169 micro-autoradiography, 172 biotin, 159 immunohistochemistry, 152 reaction direct, 158 indirect, 158

416

peroxidase, 163 pre-embedding technique ultrathin section, 230 vibratome section, 222 Vitamin H, see also Biotin

W Washes light microscopy, 143 pre-embedding method, ultrathin section, 229 vibratome section, 219 problems, 275 temperature, 144 Water DEPC, 355 sterile, 355