In Situ Hybridization: Laboratory Companion

In Situ Hybridization Edited by Melody Clark

In Situ Hybridization Laboratory Companion

Edited by Melody Clark

WILEY-VCH Verlag GmbH & Co. KGaA

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Bibliographic information published by the Deutsche Nationalbibliothek Die Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at .

 1996 Chapman & Hall GmbH, Weinheim  2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Printed in the Federal Republic of Germany Printed on acid-free paper ISBN: 978-3-527-30885-9

Forward

In situ hybridization as a technique has evolved greatly since the first published results of Pardue and Gall in 1969.Then the ability to localise crude satellite DNA preparations to nuclei was regarded as revolutionary, but now, just over 25 years later, the multiple localisation of single copy sequences onto extended nuclear fibres is routine. During this period, what was initially seen as a minor extension to t raditiona I cytogenetics has evolved into a muItid isciplina ry technique providing a link between many different areas of biological research. As an example, interphase cytogenetics using genomic in situ hybridization and chromosomal in situ supression (see chapters 4 and 1) has impacted heavily on the field of cancer genetics. Diagnosis of the progression of a disease is no longer reliant upon obtaining metaphase chromosome spreads, chromosome paints can highlight individual chromosomes within the nucleus, defining any genome changes which have taken place. Combining this with immunocytochemistry (chapter 4) enables such genome changes to be precisely correlated with cell type, allowing a further refining of diagnosis and ultimately leading to more accurate and successful treatments. The use of genomic in situ hybridization is not solely restricted to this one field, it can also reveal important clues as to evolutionary relationships between species and the spatial organisation and behaviour of chromosomes within the nucleus. All of which are ably demonstrated in chapter 1 with its agricultural bias. At the opposite end of the molecular scale, multiple single copy clones can now be routinely mapped onto extended nuclear fibres at resolutions of up to 5Kb (chapter 2). Thus providing a valuable link between all the sequencing, cloning and genetic linkage data generated by the Human Genome Project. PCR, that universal tool of molecular biology has also been adopted by in situ hybridization. As with molecular biology, it can be used as an alternative to traditional cloning and labelling strategies. Chapter 3 extols the virtues of such an adaptable technique. It is important to realise that in situ hybridization is not only limited to chromatin. Another expanding field is that of developmental genetics which is now heavily reliant upon this technique as a means for localising expression of genes, both at the organism and organ level (chapter 5). It is providing answers to not only gene product localisaForward

tion, but also in tandem with studies of transgenics, answers as to the control elements necessary for that expression in the first place. Finally we cover the least publisized level of the technique: the EM level. It must be remembered that not all answers are gained with bright multicoloured painted chromosomes or embryos, the ultimate definition of DNA and RNA localisation with respect to the ultrastructure of the cell is only obtained at the EM level, a field which has been comparatively neglected, but which shows great potential. So the aim of this manual was not necessarily to catalogue the latest in situ hybridization techniques, but to put together a whole range of techniques which reflect maior areas of current research and interdisciplinary approaches. All have proven reproducibility with immediate and practical applications. Detailed methods are supplemented with helpful hints on technique and rounded off by comprehensive troubleshooting sections (although it is hoped that this final section is not needed!) to ensure that the novice researcher gets the best introduction to this field. One should be aware that in situ hybridization is a rapidly evolving field (with an equally rapidly expanding number of acronyms!) and that this manual can only hope to provide a sound basis for interesting developments to come. Good Luck! February 1996

Forward

Melody Clark

A remark on the layout of this book: In order to facilitate the use of this book as a methodological source for your bench work, a wide page format has been chosen. Due to its proper, durable binding the book has the advantage of lying flat on your bench top for convenient use. In addition, a wide margin leaves room for your own notes and provides some key notes and pictograms that shall assist you in finding the relevant information: a pipette symbol marks the start of a step by step protocol section, a grey bar follows the whole protocol section

this symbol draws your attention to potential hazards and safety suggestions

A

comments on the key steps in methodology are highlighted by a key symbol

a “good idea” symbol marks useful hints for optimization of methodology

this pictrogram indicates discussions of alternative approaches

the tool indicates troubleshooting guides that shall help you in finding out what could or did go wrong and in solving and avoiding problems

suggestions for monitoring quality and reliability of the experimental procedure are highlighted by the magnifying glass

Layout Features

Contents

.

CHAPTER 1 Genomic In Situ Hybridization for Whole Chromosome and Genome Analysis K. ANAMTHAWAT.JONSSON. J . S. HESLOP.HARRlS0N. andT. SCHWARZACHER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 1.2 1.2.1 1.2.2 1.2.3 1.2.4 1.2.5 1.2.6 1.3 1.3.1 1.3.2 1.4 1.4.1 1.4.2 1.4.3 1.5 1.5.1 1.5.2 1.5.3 1.5.4 1.6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses of Genomic In Situ Hybridization . . . . . . . . . . . . . . . . Genome Organization of Different Sequence Classes . . . Species Identity, Differentiation and Relatedness . . . . . . . . Genomic Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spatial Organization of Chromosomes . . . . . . . . . . . . . . . Chromosome lntrogression and Rearrangements . . . . . . . Chromosome Behaviour and Meiosis . . . . . . . . . . . . . . . . . The Principle of Genomic In Situ Hybridization . . . . . . . . . . Genomic Probing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Blocking and Competitive Hybridization . . . . . . . . . . . . . . . Materials and Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . Buffers and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemicals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genomic In Situ Hybridization: Protocol . . . . . . . . . . . . . . . Chromosome Preparations . . . . . . . . . . . . . . . . . . . . . . . . . Probe DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Blocking DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Situ Hybridization Procedure . . . . . . . . . . . . . . . . . . . . . . Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 2 2 3 5 6 7 8 9 9 10 12 12 13 13 14 14 14 15 15 18

.

CHAPTER 2 Fluorescence In Situ Hybridization: Applications in Gene Mapping and Clinical Diagnostics

G. A . EVANS and L. SELLER1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 2.2 2.2.1 2.2.2 2.2.3 2.3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and Chemicals. . . . . . . . . . . . . . . . . . . . . . . . . . . Buffers and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemicals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FISH Using Single Copy Cosmid Probes . . . . . . . . . . . . . . .

25 25 28 28 30 30 31 Contents

2.31 Preparation of Metaphase Chromosomes . . . . . . . . . . . . . 2.3.2 Preparation of Metaphase Chromosome Slides for Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3 Preparation and Labelling of Probes. . . . . . . . . . . . . . . . . . 2.3.4 Hybridization of Cosmid Probes . . . . . . . . . . . . . . . . . . . . . 2.4 Multicolor FISH Using Cosmid Probes . . . . . . . . . . . . . . . . 2.41 Labelling of Probes and Hybridization Reactions . . . . . . . . 2.5 lnterphase FISH Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1 Preparation of lnterphase Nuclear Slides from Short Term Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.2 Direct Preparation of lnterphase Spreads. . . . . . . . . . . . . . 2.5.3 FISH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 FISH Analysis Using YAC Clones . . . . . . . . . . . . . . . . . . . . . 2.6.1 Preparation of Metaphase Chromosomes . . . . . . . . . . . . . 2.6.2 Isolation of YAC DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.3 Biotin Labelling of YACs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.4 FISH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.5 Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Direct Visual In Situ Hybridization (DIRVISH). . . . . . . . . . . . 2.7.1 Preparation of Stretched DNA . . . . . . . . . . . . . . . . . . . . . . 2.7.2 FISH Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7.3 Detection and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7.4 Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

31 31 32 32 33 33 34

35 35 35 36 36 36 37 37 38 38 39 39 39 40 40

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CHAPTER 3 Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ labelling (PRINS): Two Alternatives to Traditional In Situ Hybridization Methods J . HINDKJBR. C. TERKELSEN. S . K0LVRAA. J . KOCH. and L.BOLUND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials and Chemicals . . . . . . . . . . . . . . . . ........ 3.2.1 Buffers and Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2 Chemicals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 DNA-PRINSwith Oligonucleotide Probes. . . . . . . . . . . . . . 3.4 PRINS with Ddel Digested Cloned Probes . . . . . . . . . . . . . 3.4.1 Generation of Primers from Cloned Alpha Satellite DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2 PRlNS Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Multicolour-PRINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 PRINS-painting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 PRINS-PCR and Repeated-PRINSof DNA . . . . . . . . . . . . . 3.7.1 Chromosome Spreads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2 Pretreatment of Chromosome Spreads . . . . . . . . . . . . . . . . 3.7.3 Labelling Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 PRINS-PCR of mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Visualization of Hapten-Labelled Nucleotides . . . . . . . . . . Contents

45 45 47 47 47 48 48 50 52 52 53 54 55 56 57 57 58 61

3.9.1 3.9.1.1 3.9.1.2 3.9.2 3.9.2.1 3.9.2.2 3.9.2.3 3.9.2.4 3.10

Standard Slides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Digoxigenin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Micro-slides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection of Labelled DNA . . . . . . . . . . . . . . . . . . . . . . . Detection of Labelled RNA . . . . . . . . . . . . . . . . . . . . . . . Alkaline Phosphatase Visualization . . . . . . . . . . . . . . . . . Fluorescent Visualization . . . . . . . . . . . . . . . . . . . . . . . . . Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61 61 62 63 63 63 63 64 64

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CHAPTER 4 Fluorescence lmmunophenotyping and lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION): Combined In Situ Hybridization and Fluorescence lmmunophenotyping

K. WE B E R- MATTHIES EN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.1 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.3 4.3.1 4.3.2 4.3.3 4.4 4.5 4.5.1 4.6 4.6.1 4.7 4.8 4.9 4.10

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Application of the FICTION Method . . . . . . . . . . . . . . . . Defining the Cell Type of Chromosomally Aberrant Clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Defining the Differentiation State of Chromosomally Aberrant Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interpretation of Ambiguous Cytogenetic Findings . . . . . An Aid to Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overcoming the Detection Limit of FISH . . . . . . . . . . . . . Materials and Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . Buffers and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preparation of Slides for FICTION. . . . . . . . . . . . . . . . . . FICTION-Protocol 1 : lmmunophenotyping Using One Antigen Plus In Situ Hybridization of Two Centromeric Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control Studies: Specificity of lmmunophenotyping . . . . FICTION-Protocol 2: lmmunophenotyping (Two Different Antigens) Plus In Situ Hybridization (One Centromeric Probe). . . . . . . . . . . . . . . . . . . . . . . . . Control Studies: Specificity of the lmmunophenotyping . . FICTION-Protocol 3: Detection of Structural Chromosome Aberrations Within lmmunophenotyped Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . Documentation of FICTION-Results . . . . . . . . . . . . . . . . Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67 67 69 69 70 70 71 72 73 73 74 74 75 76 78 80 83 84 85 88 88

Contents

.

CHAPTER 5 In Situ Hybridization to Embryo Whole Mounts and Tissue Sections: mRNA Detection and Application to Developmental Studies T. JOWETT. M. MANCERA. A . AMORES. and Y. YAN . . . . . . . . . 5.1 5.2 5.2.1 5.2.2 5.2.3 5.3 5.3.1 5.3.2 5.4 5.4.1 5.4.2 5.4.3 5.4.4 5.4.5 5.4.6 5.4.7 5.4.8 5.5 5.5.1 5.5.2 5.5.3 5.5.4 5.5.5 5.6 5.6.1 5.6.2 5.6.3 5.6.4 5.6.5 5.7

91

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Materials and Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . 92 Buffers and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 General Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 RNase-free Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 RNA Probe Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Whole Mount In Sifu Hybridization . . . . . . . . . . . . . . . . . 97 Fixation and Pretreatments. . . . . . . . . . . . . . . . . . . . . . . . 97 Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Post-HybridizationWashes . . . . . . . . . . . . . . . . . . . . . . . 101 Incubation with Antibody . . . . . . . . . . . . . . . . . . . . . . . . . 101 Preabsorption of Antibodies . . . . . . . . . . . . . . . . . . . 102 Post-AntibodyWashes and Histochemistry . . . . . . . . . . . 103 Performing Experiments with Many Different Batches of Embryos or a Large Number of Probes. . . . . . . . . . . . 104 Embedding and Sectioning Whole Mount Embryos . . . . 105 Double-Labelling Whole Mount In Situ Hybridization . . . 105 Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Preabsorption of the Antibodies . . . . . . . . . . . . . . . . . . . 106 Alkaline Phosphatase/horseradish Peroxidase Staining (Blue/Brown). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Sequential Alkaline Phosphatase Staining (Blue/Red). . . 110 Special Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . 112 In Situ Hybridization to Tissue Sections . . . . . . . . . . . . . . 113 Preparing TESPA-Treated Slides . . . . . . . . . . . . . . . . . . . . 113 Fixation and Embedding Embryos or Tissue . . . . . . . . . . 114 Cutting Paraffin Sections . . . . . . . . . . . . . . . . . . . . . . . . . 115 Pretreatment and Hybridization of Sections . . . . . . . . . . 116 Washing of Sections and lmmunodetection of Probe . . . 118 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

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CHAPTER 6 In Situ Hybridization at the Electron Microscopic level F. WACHTLER. C. SCHOFER. and K . WEIPOLTSHAMMER . . . . . 123 6.1 6.2 6.2.1 6.2.2 6.2.3 6.3 Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . Buffers and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Labelling Nucleic Acids for In Situ Hybridization . . . . . . .

123 124 124 125 125 126

6.4 6.4.1 6.4.2 6.4.3 6.5 6.6 6.7 6.71 6.7.2 6.7.3 6.7.4 6.7.5 6.8

Preparing Tissue for Electron Microscopic In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Embedding. Cutting and Mounting . . . . . . . . . . . . . . . . . Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Situ Hybridization at the EM Level for DNA Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Situ Hybridization at the EM Level for RNA Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection of Hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gold Labelled Antibodies . . . . . . . . . . . . . . . . . . . . . . . . Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Use of the Peroxidase Reaction to Detect Hybrids at the EM Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evaluation and Interpretation of Results . . . . . . . . . . . . .

126 127 128 129 130

133 134 135 136 138 139 140 144

Appendix I Labelling DNA Probes for In Situ Hybridization . . . . . . . . . . . . . . 147 Appendix II Quantification of Efficiency of Incorporation of Labelled Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155

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

159

Subjectlndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

163

Contents

Abbreviations The following is a list of the most common abbreviations used throughout the book.

AMCA

7-amino-4-methylcoumarin-3-acetic acid-N-hydroxysuccinamide ester

AP

alkaline phosphatase

ATP

adenosine triphosphate

BClP

5-bromo-4-chloro-3-indolyl-phosphate,also known as X-phosphate

bP

base pairs

BSA

bovine serum albumin

cDNA

complementary DNA

CGH

comparative genomic hybridization

CHAPS

3-[ (chloramidopropyl)-dimethylammonio]-1 -propanesulphonate)

ClSS CTP

chromosomal in situ suppression

CY3

surprisingly this is a chemical name, not an abbreviation

DAB

diaminobenzidine

DABCO

1,4-diazabicyclo [2.2.2] octane

DAPl

4,6-diamidino-2-phenylindole

dATP

deoxyadenosine triphosphate

dCTP ddATP ddCTP ddGTP ddTTP

deoxycytidine triphosphate dideoxy cytidine triphosphate

DEPC

diethylpyrocarbonate

dGTP

deoxyguanidine triphosphate

DIG

digoxigenin

cytidine triphosphate

dideoxy adenosine triphosphate dideoxy guanidine triphosphate dideoxy thymidine triphosphate

Abbreviations

Abbreviations

DIRVlSH

direct visual in situ hybridization

DMF

dimethyl formamide

DMSO

dimethyl sulphoxide

DNA

deoxyribonucleic acid

DTT

dithiothreitol

dTTP

deoxythymidine triphosphate

dUTP

deoxyuridine triphosphate

E DTA

ethylene diaminotetraacetic acid

EGTA

ethylene glycol tetraacetic acid

EM

electron microscope/electron microscopic

FACS

fluorescence activated cell sorting

FISH

fluorescent in situ hybridization

FlTC

f Iuoroscene isothiocyanute

GTP

guanidine triphosphate

Kb

kilobase pairs

mRNA

messenger ribonucleic acid

N-AS-MX

napthol As-Mx phosphate

NBT

4-nitro blue tetrazolium chloride

PBS

phosphate buffered saline

PCR

polymerase chain reaction

POD

horseradish peroxidase

POX

peroxidase

PVP

polyvinylpyrrolidone

rDNA

ribosomal deoxyribonucleic acid

RNA

ribonucleic acid

rt/RT

room temperature

SDS

sodium dodecyl sulphate

SDW

sterile distilled water

ssc

standard saline citrate

TESPA

3-aminopropyltriethoxysilane

tRNA

transfer ribonucleic acid

UTP

uridine triphosphate

uv

ultraviolet

YAC

yeast artificial chromosome

list of Contributors

Amores, A., Department of Biochemistry and Genetics, The Medical School, The University, Newcastle upon Tyne, NE2 4HH, UK Anamthawat-Jonsson, K., Agricultural Research Institute, Keldnaholt, Reykjavik, IS-112, Iceland Bolund, L., Institute of Human Genetics, Aarhus University, DK-8000 Arhus C, Denmark Evans, G. A., McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, Texas 75235-8591, U.S.A Heslop-Harrison, J. S., Karyobiology Group, John lnnes Centre, Colney Lane, Norwich, NR4 7UH, UK Hindkizer, J., Institute of Human Genetics, Aarhus University, DK-8000 Arhus C, Denmark and Danish Cancer Society, Department of Cytogenetics, Tage Hansens Gade 2, DK-8000 Arhus C, Denmark Jowett, T., Department of Biochemistry and Genetics, The Medical School, the University, Newcastle upon Tyne, NE2 4HH, UK Koch, J., Institute of Human Genetics, Aarhus University, DK-8000 Arhus C, Denmark/Danish Cancer Society, Department of Cytogenetics, Tage Hansens Gade 2, DK-8000 Arhus C, Denmark K~llvraa,S., Institute of Human Genetics, Aarhus University, DK-8000 Arhus C, Denmark Mancera, M., Department of Biochemistry and Genetics, The Medical School, The University, Newcastle upon Tyne, NE2 4HH, UK Schofer, C., Histologisch-embryologisches lnstitut der Universitat Wien, SchwarzspanierstraBe 17, A-1090 Wien, Austria Schwarzacher, T., Karyobiology Group, John lnnes Centre, Colney Lane, Norwich, NR4 7UH, UK Selleri, L., Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5324, U.S.A Terkelsen, C., Institute of Human Genetics, Aarhus University, DK8000 Arhus C, Denmark

List of Contributors

Wachtler, F., Histologisch-embryologisches lnstitut der Universitat Wien, SchwarzspanierstraBe 17, A-1090 Wien, Austria Weber-Matthiesen, K., lnstitut fur Humangenetik, Klinikum der Christian-Albrechts-Universitat zu Kiel, Schwanenweg 24, 24105 Kiel, Germany Weipoltshammer, K., Histologisch-embryologisches lnstitut der Universitat Wien, SchwarzspanierstraBe 17, A-1090 Wien, Austria Yan, Y., Department of Biochemistry and Genetics, The Medical School, The University, Newcastle upon Tyne, NE2 4HH, UK

List of Contributors

In Situ Hybridization Melody Clark Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Genomic In Situ Hybridization for Whole Chromosome and Genome Analysis K . A NAMTHAWAT-J 0NSSON, J .S. HESLOP- HARR ISON, and T. SCHWARZACHER

1.1 Introduction Genomic in situ hybridization is a method involving hybridization of labelled total genomic DNA, used as a probe, to spread or sectioned denatured chromosome preparations in situ, followed by detection and visualization of the sites of hybridization. The earliest DNA:DNA hybridization experiments (e.g. Pardue and Gall 1969, 1970) demonstrated that genomic DNA of different species, such as mouse and the toad Xenopus, essentially showed no hybridization to one another, and the studies were able to localize different DNA fractions within the same species. The species-specificity of genomic DNA hybridization was utilized many years later to study mammalian chromosomes in hybrid cell lines and nuclear organization (Manuelidis 1985; Pinkel et a/. 1986) and also plant chromosome and nuclear organization (Heslop-Harrison et a/. 1988; Schwarzacher et a/. 1989). Recently, the technique has been modified for studies of chromosome evolution in both cereals (Anamthawat-Jonsson etal. 1991 and Heslop-Harrison 1992; Leggett et a/. 1994; Orgaard and HeslopHarrison 1 9 9 4 ~ b)~and mammals (Houseal et a/. 1995; Scherthan et a/. 1994), and also for application to chromosome analysis of solid tumours and leukaemias (see chapter 4 ) . Such modifications, including reverse chromosome painting (Carter etal. 1992; Joos etal. 1993) and comparative genomic hybridization (Kallioniemi et a/. 1992; du Manoir et a/. 1993),allow detection of copy number changes in cancer throughout the whole genome without prior knowledge of the aberrations. In plants, the genomic in situ hybridization was first described for use in identifying parental species in cereal hybrids and cultivars (Schwarzacher etal. 1989; Le etal. 1989).The technique was further investigated in the latter by Southern blot analysis where the specificity could be regulated by using either competitive hybridization or blocking approaches (Anamthawat-Jonsson et a/. 1990). Genomic in situ hybridization has now become an essential tool in molecular cytogenetic studies of a wide range of species. This chapter aims to review both its applications in biological research at the genome and whole chromosome level and its practical uses in genome analysis and plant breeding. A detailed outline of

definition

history species-specifity of genomic DNA

chromosome modifications

analysis of hybrid genome

M. Clark (Ed.), In Situ Hybridization.0 Chapman & Hall, Weinheim. ISBN 3-8261-0038-7

the principles involved is followed by a selected protocol along with comprehensive notes suggesting possible optimization and troubleshooting. The extensive reference list includes sources of specialized protocols. The methods described have been used routinely with plant chromosomes and are chosen on the basis of excellent reproducibility and simplicity.

1.2 Uses of Genomic In Situ Hybridization applications

Over the last five years, numerous laboratories have successfully instigated in situ hybridization projects. Many of these have used genomic in situ hybridization. There are a large number of potential applications of this technique for investigating nuclear structure and nuclear interactions, these are reviewed below :

1.2.1 Genome Organization of Different Sequence Classes The genome of a eukaryotic organism comprises all the genes and non-coding DNA in a complete haploid nucleus. Genomes differ between species in size, organization and nucleotide base sequence. Overall size, measured in base pairs (bp), shows a huge range between species (Szathmary and Maynard Smith 1995; Bennett and Smith 1976):for example 14 M b p (14x lo6 bp) in yeast Saccharomyces, 150 M b p in the plant Arabidopsis thaliana, 3,500 M b p in human, 17,000 M b p in bread wheat, and some 130,000 Mbp in the lily Fritillaria and lungfish Protopterus.The significance of genome size variation between species, and the reasons for the relative stability within species, are largely unknown. Some sequences are highly conserved and hence shared by evolutionary related species, while others have evolved rapidly and become essentially species- or genome-specific sequences. It is possible to identify many of these sequences by genomic probing on Southern blots, where common sequences can be blocked by means of competitive hybridization, and therefore revealing species- or genome-specific DNA (Anamthawat-Jonsson and Heslop-Harrison 1993). The reannealing kinetics of denatured DNA fragments from total genomic (nuclear) DNA of 23 species of higher plants were measured and compared with the nuclear DNA mass (Flavell et al. 1974).This showed that most of the variation in DNA content was due to differences in middle repetitive and highly repetitive DNA - typically sequence motifs between 2 and 10,000 b p long that are repeated tens to hundreds of thousands of times in the genome. The organization of these sequences within the genome is an important aspect of many biological studies and genome mapping. K. ANAMTHAWAT-J6NSSON et al.

In this context, genomic in situ hybridization is able to provide important information including the physical locations of different sequence classes with respect to the centromere, telomeres and chromosome arms. The physical localization is, in turn, providing insights into mechanisms of sequence distribution. Some genome-specific sequences are localized in heterochromatic blocks and are long tandem arrays of repetitive DNA e.g. (Vershinin eta/. 1994; Kamm et a/. 1994; Schmidt eta/. 1991).These are typical of the 'satellite DNA' class found in many species, DNA characterized by its generally simple molecular organization that is often clustered at a small number of sites on single, multiple or all chromosomes of a given genome. The chromosomal localization suggests that such satellite sequences have possibly evolved by means of sequence amplification and deletion through unequal crossing-over (Charlesworth et a/. 1994).Other genome-specific sequences are dispersed throughout the whole genome, some of which are likely to be in the class of retrotransposons, either active or degenerated (Flavell et a/. 1992; Smyth et a/. 1989). Furthermore, several studies of these elements showed their genomic organization is non-random (Moore et a/. 1991) with the sequences concentrated in the distal half of chromosome arms (Suoniemi eta/. 1996))similar to that of the recombination sites or the gene regions (Moore et a/. 1993), or with exclusion from centromeric, telomeric and NOR parts of chromosomes (Schmidt et a/. 1995; Pearce et a/. 1995).The study of the organization of various components of genome-specific DNA will contribute to the understanding of genome organisation, species relationships and to the integrity of a genome.

physical mapping

sequence distribution

satellite DNA

retrotransposons

1.2.2 Species Identity, Differentiation and Relatedness One of the most direct applications of genomic in situ hybridization is in the establishing of species relationships. One may need to examine the extent of genetic relationships between closely related species for the purposes of resource management or assessment of biodiversity. It is also important to verify commercial clones and cultivars used in agriculture and forestry, to detect pathological diseases, and to evaluate evolutionary changes at the molecular and organism levels. Where interspecific and intergeneric hybridization forms an important part of evolution, it is often necessary to determine the ancestral origins of polyploid species, or the origin of alien chromosomes or chromosome segments in natural and cultivated hybrid derivatives. In plants, genomic in situ hybridization has been used successfully with a wide range of species, including Brassica, crocus, citrus, potato, tobacco and cereal species (see Heslop-Harrison and Schwarzacher 1993). In clinical studies, the approach was used, at first, for the screening of interspecific hybrid cell lines for retained human chromosomes and to detect chromosomal rearrangements, but is now modified and has become an essential tool in the field of cancer cytogenetics.

establishing species relationships

Genomic In Situ Hybridizations for Whole Chromosomeand Genome Analysis

Fig. 1 (A-F). Fluorescent photomicrographs of plant mitotic chromosomes after genomic in situ hybridization. Scale bar: 10pm. (A) Two-colour in situ hybridization of metaphase chromosomes of a wheat x lymegrass hybrid (Triticum aestivum x Leyrnus arenarius) showing red fluorescence of rhodamine labelled wheat and green fluorescence of fluorescein labelled lymegrass chromosomes. No unlabelled blocking DNA was used. (B) Chromosomes at late mitotic anaphase from a barley hybrid (Hordeurnvulgare x H. bulbosum)showing lagging yellow-green labelled H. bulbosurn chromosomes with the morphology indicating poor centromeric activity. Propidiurn iodide stained H. vulgare chromosomes show normal mitotic behaviour. Genomic in situ hybridization differentiates the different barley genomes after blocking 40x. (C) lnterphase cells from a wheat x lymegrass arnphiploid showing individual (green) lymegrass chromosome domains. (D) lnterphase and prophase cells of wheat containing an added chromosome arm from a wild barley species (H. chilense).The alien chromosome arm also shows territorial organization at interphase. (E) The metaphase chromosome of the same wheat in the previous figure showing the translocation between the green labelled barley chromosome arm and the propidium iodide stained wheat arm. Unlabelled wheat DNA was used as a block at 15x the probe amount. (F) Metaphase chromosomes of a grass hybrid (Festulolium) showing several intergenomic translocations (kindly provided by Dr lzolda PaSakinskiene).Loliurn DNA was used as a probe (fluoresced yellow-green) and Festuca as a block

K. ANAMTHAWAT-J6NSSON etal.

Fig 1A shows an application of genomic in situ hybridization in identifying the species origin of genomes in an artificial cereal wide hybrid: bread wheat (Triticum aestivum) was cross-hybridized with perennial lymegrass (Leymus arenarius) for production of lines with improved characteristics ( Ana mt hawat-J onsson et al. 1996). Hybridization between different species or genera is a common practice in plant breeding programmes aiming to expand the crop genetic base. Numerous useful traits have been transferred from the wild species into crops via such hybrids, and these include characters contributing to disease resistance and tolerance to environmental stress. Here, genomic in situ hybridization has proven to be a valuable tool for detection of such transfers, especially when it is important to control the size of chromosome segments or to identify the breakpoints of the new chromosomal rearrangements (Heslop-Harrison et al. 1990; Schwarzacher et al. 1992;Thomas et al. 1994).Chromosomal transfers of useful characters can also have disadvantages, for example gene loss due to chromosomal substitution, karyotypic instability resulting in non-uniform genetic stocks and meiotic irregularities leading to fertility reduction. It is therefore important to be able to characterize genomic recombination accurately, and this method is now being used to characterize a series of small alien chromosome segments transferred into wheat from Aegilops umbellulata (Castilho et al., in preparation). lnterspecific hybridization is a known evolutionary event that is able to secure the species survival and adaptability in a changing environment. For example, postglacial expansion and genome subdivision in the European grasshopper is thought to have been facilitated by hybrid zones acting as barriers to gene flow (Hewitt 1988; Cooper et al. 1995). In many plant species, the gene flow, especially between species by means of introgressive hybridization, has selective advantages because it provides new genotypes that can tolerate environmental stresses (Bloom and Lewis 1972; Anamthawat-Jonsson 1994). When interspecific hybridization is followed by polyploidization, a speciation can occur. Thousands of plant species are natural allopolyploids. Genomic in situ hybridization has been used to reveal such intergenomic relationships: for example in wheat (Mukai et al. 1993; Jiang and Gill 1994), oat (Leggett et al. 1994),tobacco (Kenton et al. 1993) and crocus (Orgaard et al. 1995).Relationships between different genomes are complex. The gene flow between co-existing genomes can occur via somatic or meiotic recombination, but it is also prevented through a stringent genetic control in order to maintain the genome stability. The understanding of genomic relationships may also reveal an important mechanism or pattern of gene expression in vivo in living organisms.

detection of chromosome transfers

monitoring of interspecific hybridization

1.2.3 Genomic Stability The stability of a genome is presumably under strict genetic control as it is essential for the survival of a species and the functional integrity of an organism. Genomic instability detected in human breast carcino-

monitoring of diseaserelated genomic instability

Genomic In Situ Hybridizations for Whole Chromosome and Genome Analysis

monitoring of genome stability in interspecific hybrids

mas, for example, has been associated to mutations in genes controlling the cell cycle such as the p53 tumour suppresser gene (Eyfjord et al. 1995; Hollstein et a/. 1991). The instability occurs in many forms: allelic loss, DNA amplification and chromosomal rearrangements (Steinarsdottir et a/. 1995; Anamthawat-J6nsson et al. 199510). This complexity makes it very difficult to identify any primary causes leading to the diseases. Therefore the increasing research effort is to gain an overall assessment of genomic instability in such complex solid tumours, especially by using the comparative genomic hybridization (CGH) method (Kallioniemi et al. 1992).This method, which is based on a competitive and simultaneous hybridization of tumour and normal DNA to normal chromosome spreads, is able to detect changes in copy number (gains and losses). It is more sensitive in the detection of amplifications compared to deletions. It cannot detect balanced chromosome rearrangements and no information is available on the ways gains and losses are arranged within marker chromosomes. Nevertheless, it is very important to draw detailed profiles of these many complex tumours before any specific changes can be examined and meaningful results obtained. lnterspecific hybrids have been used as models to study aspects of genomic relationships and stability. Genome instability may include any deviant behaviour producing abnormal structure or karyotype, whether or not it leads to genome or chromosome elimination (Bennett 1981). In hybrid cell lines of human and murine origin, human chromosomes tend to be eliminated, giving rise to cell lines with retained individual human chromosomes that can be isolated to make chromosome-specific DNA libraries. In plants, a barley hybrid (Hordeurn vulgare x H. bulbosurn), examined by electron microscopy (Schwarzacher et a/. 1992) and genomic in situ hybridization (Anamthawat-Jonssonet a/. 1993), revealed not only that genomes of different parental origins did not intermix but also that the stable H. vulgare genome had larger centromere-associated structures and the chromosomes behaved more normally during cell divisions than when compared with the less stable H. bulbosurn genome. The H. bulbosurn chromosomes tended to be peripheral to the metaphase plate, lagged behind during anaphase and telophase (Fig. 1B) and were not included in the daughter cells. Genomic in situ hybridization showed that micronuclei included H. bulbosurn origin chromosomes only. In plants, genomic instability can have adverse effects on the phenotype and such defects in the germ line can cause reduced fertility due to pairing failure at meiosis.

1.2.4 Spatial Organization of Chromosomes monitoring the spatial organization of chromosomes

Accumulating evidence suggests that nuclear function is closely related to three-dimensional structure. Hence fluorescent in situ hybridization has become an essential tool for the examination of spatial organization within the nucleus (Cremer et a/. 1993; HeslopHarrison et a/. 1993).The distribution of telomeres, centromeres and

K. ANAMTHAWAT-J6NSSON et a/.

chromosome-specific subsatellite domains in cultured mouse lymphocyte nuclei have been shown to be cell cycle dependent (Vourc'h et al. 1993). The higher level organization of transcription, splicing and transport involving several RNA classes is being studied with a combination of in situ hybridization, immuno-fluorescence and digital imaging microscopy (Carter et al. 1993; Xing et al. 1993).Spatial organization of chromosomes has been studied in both plant and animal nuclei with the view of gaining insight into the relationships between nuclear architecture and function (Heslop-Harrison et al. 1993; Cremer et al. 1993).Genomic in situ hybridization has contributed significantly towards such studies. The idea of territorial organization of chromosomes was suggested at the turn of this century. Rabl (1885)described a model of interphase structure where centromeres tended to be positioned at one end of the nucleus and telomeres at the opposite end. Subsequent studies have shown the Rabl-orientation in several cell types (e.9. Cremer et al. 1982; Anamthawat-Jonsson and Heslop-Harrison 1990), while other studies showed non-Rabl compartmentalization of chromosome domains (Manuelidis and Borden 1988) or random spatial localization of genes (Montiin et al. 1994).Genomic in situ hybridization has consistently shown territorial organization of individual chromosomes (Fig. 1C) or sub-chromosomal regions (Fig. 1D) in the interphase nuclei of both plant and animal species. However, the spatial positioning in the nucleus may be dynamic and that may be associated with cell differentiation and nuclear function. The compartmental organization of chromosome itself should indicate a maior significance. Cremer et al. (1993) proposed a model predicting that the surfaces of chromosome territories and a space between them provide a network-like three-dimensional nuclear compartment for gene expression, mRNA splicing and transport.

1.2.5 Chromosome lntrogression and Rearrangements Chromosome rearrangements can occur in somatic or reproductive cells, in culture or in living organisms, spontaneously or under external influences. Chromosomal rearrangement is probably a common event in evolution, and can provide a means for gene transfer and introgression. Certain rearrangements are neutral if they do not present immediate disadvantages or benefits, and they do not disrupt the genomic integrity. Some translocations, especially the Robertsonian type, are maintained in particular populations of animals or plants without affecting either the phenotype or fertility of the individuals. Whether or not they may have a selective advantage at some point in evolutionary time is difficult to determine, however, the changes are not uncommon in nature. Mutational changes have often been detected in artificial conditions, for example karyotype instability in wheat cell suspension cultures (Leitch et al. 1993) and interspecies chromosome rearrangements in human-hamster hybrid cell lines (Pinkel et al. 1986).

monitoring of chromosome rearrangements

Genomic In Situ Hybridizations for Whole Chromosome and Genome Analysis

In situations where chromosomes are derived from different genomic origins, genomic in situ hybridization has proven to be a valuable tool in the detection of such changes. lntergenomic translocations have been observed, for example, in the cultivated allotetraploid wheat (Jiang and Gill 1994) and in the grass hybrid Psathyrostachys x Leymus (K Anamthawat-Jonsson and M 0rgaard, unpubl). In the wide hybrids and derivatives made during plant breeding, genomic in situ hybridization is used routinely to detect chromosome transfers and rearrangements in cereals (Fig. 1 E; Schwarzacher et al. 1992~). It has detected somatic translocations in potato (Wilkinson et al. 1995), which was not thought to occur during the process of dihaploid induction. Furthermore, some groups of species seem to tolerate impressively high frequencies of intergenomic recombination, for example, fescue and ryegrass hybrids (Fig. 1F; I PaSakinskiene and K Anamthawat-Jonsson, unpubl; Thomas et al. 1994).The studies of gene flow and introgression have become more accurate as the use of genomic in situ hybridization has been extended to suit a wider range of species.

1.2.6 Chromosome Behaviour and Meiosis studying of chromosome pairing at meiosis

gameto- and sporogenesis

Genomic in situ hybridization can reveal aspects of meiosis such as chromosome pairing, synapsis, meiotic segregation and pre- or postmeiotic behaviour of chromosomes. In cereal breeding programmes, for example, where gene transfers from related crop or wild species have been manipulated cytogenetically, it is necessary to be able to assess and identify specific pairing of homeologous chromosomes by which intergenomic recombination can occur (Le and Armstrong 1991; Miller et al. 1994; Schwarzacher and Heslop-Harrison, 1995). This type of introgression is particularly difficult to obtain in natural allopolyploid species such as wheat, because the genetic system has evolved to prevent such pairing, and therefore the breeders have to find ways of suppressing the genetic control first (Merker 1992).Once the intergenomic pairing has been induced, recombination leading to chromosome transfer can be achieved, for example the transfer of a whole chromosome arm from a Chilean barley species into wheat provides plants with resistance to root-knot nematode (Fig. 1 E; Person-Dedryver et al. 1990). It is also important to investigate the process of gameto- or sporogenesisto assess the production of cytogenetically viable gametes. In some breeding programmes, where self-fertility of wide hybrids is extremely difficult to obtain due to high ploidy level (e.g. Anamthawat-Jonsson et al. 1996))genomic in situ hybridization has been used to identify 2n gametes since they are valuable for plant breeding using a direct backcrossing strategy. The production of unreduced gametes in tetraploid oat was recently investigated, and interestingly the cytogenetic results showed that the mode of chromosome doubling was found to be due to failure of cell wall formation during the last mitotic division that preceded meiosis (Katsiotis and Forsberg

K. ANAMTHAWAT-J6NSSON et al.

(1995).The resulting binucleate cells underwent normal meiotic divisions and formed pollen grains with an unreduced number of chromosomes. Such knowledge is useful in practical breeding. Chromosome behaviour during the first meiotic interphase and prophase has received considerable attention because any aberrations in the process can have critical inheritable effects. A high incidence of sex chromosome aneuploidy in human sperm, for example, has been suggested to result from the particular behaviour and spatial orientation of sex chromosomes in the meiotic interphase (Armstrong et al. 1994). Chromosome aberration models can also be tested, or predicted, by examining the products of meiosis, like those involving ionizing radiation induced DNA double strand breaks prior to DNA replication (Lucas and Sachs 1993). But the early meiotic stages can be examined directly by in situ hybridization, and likewise by genomic in situ hybridization, as shown with the pachytene chromosomes in surface-spread pollen mother cells of rye (Albini and Schwarzacher 1992). This has great potential for studying chromosome behaviour and meiotic mechanisms at high-resolution.

1.3 The Principle of Genomic In Situ Hybridization 1.3.1 Genomic Probing Total genomic DNA can be used as a probe for DNA:DNA hybridization on a Southern blot or to a chromosome preparation. This is referred to as genomic probing, while the phrase genomic in situ hybridization has been used specifically for in situ hybridization. Fig. 2 shows genomic probing on a Southern blot where total genomic DNA from a wild barley Hordeurn bulbosurn (Hb) was used as a probe to hybridize to Dral digested fragments of total genomic DNA from its own species (Hb), its closely related species the barley H. vulgare (Hv) and their interspecific hybrid plant ( H v x Hb). The lambda size marker lane indicates 21,5 and 2Kb. The amount of DNA loaded on gel was similar on all three lanes (approximately 1pg), but the hybridization signal is substantially different. The signal of probe hybridization is strongest on the Hb lane, weakest on the Hv lane and intermediate on the hybrid lane, as expected. In this case, unlabelled total genomic DNA from the Hv species was used as a block to suppress cross-hybridization between these closely related species, and hence the differentiation was greatly improved (see comparison of probe specificity between blocked and unblocked hybridization in Anamthawat-Jonsson et al. 1990).This Southern hybridization experiment confirms the specificity of genomic probe developed for use with genomic in situ hybridization on the Hv x Hb hybrid chromosomes (Schwarzacher et a/. 199213; Anamthawat-Jonsson et a/. 1993).

~ i2. ~~~~~d ~ . explainedin text

Genomic In Situ Hybridizations for Whole Chromosome and Genome Analysis

Total genomic DNA from closely related species consists of DNA sequences that are conserved, and hence are common factors determining effectivenessof genome discrimination

between the species, and sequences that have diverged evolutionarily ie. species-specific. The proportion of the common and species-specific sequences in a genome, however, differs between groups of species, and this determines the effectiveness of the genomic probing method. The power of the method also depends on the nature and organization of species-specific DNA sequences; not all genomes can be discriminated by this method. Among cereal species, the repetitive sequences are distributed throughout the whole genome, consisting of dispersed and localized sequences, of low and high copy number, so the genomic probing is informative and quantifiable for discriminating between closely related species. The human genome too has species-specific sequences distributed over the whole genome. But in some genera, such as Brassica, citrus, birch and perhaps those others with very small genome size, the species-specific sequences tend to be mainly localized, tandemly repeated, satellite sequences. These can provide insight into specific molecular events that may have occurred during genome evolution, but they may not prove to be effective probes for genomic in situ hybridization. Dot-blot experiments demonstrated for the first time the specificity of genomic probes to distinguish plant species (Schwarzacher ef al. 1989))bacterial strains (Yap et al. 1989) and to screen infected farm animal tissue for bacterial disease (Hopper et al. 1989).The experiments were later expanded using restriction enzyme digested DNA, where control of hybridization stringency and competitive blocking were applied and hybridization signals could be quantified (Anamthawat-Jonsson et al. 1990). Similar experiments allowed particular species-specific sequences to be identified and isolated (Cook and Sequeira 1991; Anamthawat-Jonsson and Heslop-Harrison 1993).These experiments have contributed to the understanding of genomic probing and allowed the optimization of the genomic in situ hybridization method.

1.3.2 Blocking and Competitive Hybridization stringency: relation to specifity and sensitivity

Genomic probing with the control of stringency alone is often able to distinguish different species or genera because the DNA sequences are sufficiently diverged. Low stringency (typically 75 %) gives strong signal, but may not separate all genomes or allow assessment of the similarity between the probe-origin and the genomes being probed. Increasing hybridization stringency improves discrimination, but labelling may not be as uniform and the rate of hybridization of low copy sequences is reduced (Sealey et al. 1985) along with the efficiency of hybridization of short DNA fragments (Meinkoth and Wahl 1984). In such situations, blocking of cross-hybridization using high concentrations (20-1OOx that of the probe) of unlabelled DNA from

K. ANAMTHAWAT-J6NSSON et al.

another species (typically the species not used a probe in the nuclei being examined) increases the specificity of the probes without compromising sensitivity too much. The blocking used in our genomic in situ hybridization applications is based on competitive hybridization between the labelled probe, the chromosome target and the unlabelled blocking DNA. The block is prepared as short fragments of total genomic DNA from a related species and is applied in many fold the probe amount (2x to 50x). Hence the maior effect of such blocking may be due to:

blocking improves specifity

1) Hybridization between probe DNA and the block 2) Hybridization between the block and its homologous sequences in the target

3) A combination of both of the above. Southern experiments show that the blocking is likely to occur to all types of DNA families over the entire genome. As species-specific highly repeated DNA fragments become more apparent with increased blocking, the level of intensity of low copy and middle repetitive sequences reduces. The use of blocking DNA is extensive in mammalian cytogenetics where there is a need to suppress cross-hybridization of cloned probes to non-target sites on chromosomes (Landegent et a/. 1987; Lichter etal. 1988; Pinkel etal. 1988), although for an unknown reason, considerably lower concentrations relative to probe are effective than in the plant experiments. Pre-reassociation of human cloned probes, containing dispersed repeated sequences (e.9. Alu and Kpnl families), with a large excess of sonicated total human DNA or the Cot=l fractions allows single and low copy number components to be detected, and this was referred to as chromosomal in situ suppression (CISS)hybridization (Cremer et a/. 1988).Such blocking is particularly valuable in the physical mapping of genes in large clones such as cosmids and YACs. Specificity of the standard chromosome-specific painting probes and of the reverse painting, especially those obtained by DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction; (Carter etal. 1992; Telenius etal. 1992), is also significantly improved. Furthermore, a competitive blocking hybridization between differently labelled probes has added a considerable leap in the sensitivity of the genomic probes. This was proven in the case of the comparative genomic hybridization (CGH; Kallioniemi et a/. 1992) where tumour and normal DNA was allowed to compete for hybridization with the target chromosomes. A similar approach has been used with plant hybrids and it produces positive labelling of different genomes simultaneously without using unlabelled blocking DNA (Fig. 1A; Anamthawat-Jonsson and Reader 1995).

chromosomal in situ suppression (CISS) hybridization

blocking with differently labelled probes

Genomic In Situ Hybridizations for Whole Chromosome and Genome Analysis

1.4 Materials and Chemicals 1.4.1 Buffers and Reagents Anti-digoxigenin-FITC in 5 % BSA: Dilute the stock anti-digoxigenin-FlTC (antibody raised in sheep, Boehringer Mannheim, 200 pg/pl) according to batch recommendation. Usually, 1 :9 dilution gives good results. Use 50 pl per slide, prepare fresh and keep away from light. Antifade mountant: Glycerol, Citifluor AF1 or PPD (p-phenilendiamine, 1 mg/ml in water) in either glycerol or, preferably, DABCO (1,4-di-azobicyclo-(2,2,2)-octane), diluted 1:9. Bovine serum albumin (BSA), immunoglobin free, 5 % (w/v): Prepare fresh in 2x SSC, use about 100 pI per slide. DAPl (diamidinophenylindole),1 pglml: Prepare 100 pglml stock solution in water, then dilute it 1 :lo0 in a buffer made by mixing 18 ml of 0.1 citric acid and 82 ml of 0.2 Na2HP04 dihydrate, pH 7. Store at -20 "C in aliquots. Dextran sulfate, 50% (w/v): Dissolve dextran sulfate in distilled water, heat to 70 "C to help dissolving, which may take 2 days. Filter sterilize and store at 4 "C.

A A

use fume cupboard

use fume cupboard

DNase-free RNase, 0.1 mg/ml: Dilute 10 mg/ml RNase-A stock solution with 2x SSC, prepare fresh. The stock is prepared in 10 mM Tris pH 8, 15 mM NaCI, and boiled for 15 min to inactivate residual DNase. Store at -20 "C. Formamide: Use high grade formamide, e.g. Sigma F7508 or Merck 9684. Make aliquots and store at -20 "C. The formamide concentration in hybridization buffer is between 55 and 60%. Paraformaldehyde 4 % (w/v): Add 4 g in 80 ml distilled water in a fume hood, heat "C for 10 min, add 1 or 2 drops of 1M NaOH to clear the solution and adjust the volume to 100 ml. Prepare fresh.

to 70 to 80

Propidium iodide, 2 pg/ml: Dilute 100 pg/ml stock in 4x SSC. Prepare fresh and use about 100 pI per slide.

20x SSC stock: 3 M NaCI, 0.3 M Na3 acetate, pH 7. Dilute to 2x SSC or stringency as required for use. Stringent wash solution: 50% (vlv) formamide, 2x SSC. Prepare fresh. Wash solution: 2x SSC and 4x SSC with Tween 20,2% (v/v).

K. ANAMTHAWAT-J6NSSON etal.

1.4.2 Chemicals Anti-digoxigenin (FAB fragment) conjugated to fluorescein Anti-digoxigenin (FAB fragment) conjugated to rhodamine Antifade mountant

Boehringer Mannheim

1207741

Boehringer Mannheim

1207750

Citifluor Ltd or: Vectrashield Vector Laboratories Avidin conjugated to fluorescein Vector Laboratories or: Sigma Vector Laboratories Avidin conjugated to Texas Red Boehringer Mannheim Biotin-16-d UTP Sigma BSA Cellulase (fromAspergillus niger) Calbiochem Amersham Coumarin-4-dUTP Sigma DAPl Sigma Dextran sulphate Boehringer Mannheim Digoxygenin-11-dUTP Sigma Dnase-free Rnase Amersham Fluorescein-11-dUTP Sigma Formamide Labelling enzyme mix (DNA poly- Gibco-BRL merase I and DNase I) Paraformaldehyde Sigma Pectinase [from Aspergillus niger) Sigma Sigma Propidium Iodide Amersham Rhodamine-4-dUTP Sigma Streptavidin conjugated to Cy3 Boehringer Mannheim Unlabelled nucleotides (dATP, dCTP, dGTP, dTTP)

AF-1 H1000 A2001 A2050 A2006 1388908 A7638 21947 RPN 2123 D1388 D8906 1093088 R9000 RPN 2121 F7508 18162-016 P6148 P9179 P4170 RPN 2122 S6402 1277049

1.4.3 Equipment Most equipment used in this protocol is standard for most laboratories. However, two specialised items are described below: Hybridization chamber: We use a programmable heating block modified for in situ hybridization (Heslop-Harrison et al. 1991) (e.g. from Hybaid, UK). If a heating block is not available, a chamber can be prepared from a box with lid, in which slides can be placed above pads of tissue paper soaked with water. This chamber is used floating in a heated water bath. Epifluorescent photomicroscope with filter blocks for UV (fluorescence colour blue, e.g. Leitz A), blue (fluorescence colour green, e.g. Leitz 13) and green excitation (fluorescencecolour red, e.g. Leitz N2), and fluorescent objectives 25 and 1OOx. If an epifluorescent microscope is not available, techniques parallel to those described may be used with colorimetric detction of hybridization sites mediated by enzymatic reactions.

Genomic In Situ Hybridizations for Whole Chromosomeand Genome Analysis

1.5 Genomic In Situ Hybridization: Protocol 1.5.1 Chromosome Preparations For most of our experiments we use metaphase spreads prepared by either using the squash technique or from cell suspensions, after partial digestion with cellulase and pectinase to remove much of the cell walls and cytoplasm. Plant tissue containing many dividing cells is found in root tips (1-2 cm) from young seedlings, or in new roots in pots or hydroponic culture. Alternatively, flower buds, anthers, carpels or apical meristems can be used for sources of dividing mitotic and meiotic cells. Sectioned material may also be used with success, and is particularly valuable for three-dimensional reconstructions (Leitch et al. 1991; Leitch et a/. 1990; Binder et al. 1986). The enzymatic method of plant chromosome preparations for in situ hybridization has been described in detail in Schwarzacher et al. (1989)and Schwarzacher and Leitch (1994).

@

chromosome preparation

check quality of chromosome spread

Briefly, the plant tissue (e.9. seedlings with root-tips) is pre-treated for 24 h in ice-water to collect metaphases and fixed in 3: 1 absolute ethanol and glacial acetic acid. The fixed material can be stored at -20 "C for 1-2 months. This material is then digested with 1 % cellulase and 10% pectinase in 0.01 M citric acid/sodium citrate buffer (pH 4.6). The meristematic cells were squashed in 45% acetic acid on (chromic acid cleaned) microscope slides, or a cell suspension is dropped onto the slides. The quality of the chromosome spread preparations is critical to the success of the in situ hybridization. We therefore check the slides carefully with phase contrast and select only the best ones that contain well-spread metaphases with relatively high mitotic index and the preparations are clean, thin and with no cytoplasm and cell wall. Routinely, we discard half of all batches of fixations and half of slides made from good batches of fixations.

1.5.2 Probe DNA

@

preparation of probe DNA

Total genomic DNA from a species to be used as probe for in situ hybridization is sheared to the size of 3 to 10 kb, mechanically by drawing the (RNase-free) DNA solution about 100 times through a 1 ml syringe fitted with a fine needle, or by sonication. The amount of labelled probe used per slide is 100 to 300 ng.

A protocol for labelling of total genomic DNA is described in the appendix to this book. The method can be optimized (Leitch et al., 1994) for labelling by nick translation using fluorochrome-, digoxigeK. ANAMTHAWAT-JONSSONet al.

nin-, or biotin-conjugated nucleotides. The total genomic DNA from most plant species can be isolated from fresh leaf tissue by using a standard isolation protocol or a simple CTAB (hexadecyltrimethylammonium bromide) method without caesium chloride gradient extraction (Doyle and Doyle, 1990). The incorporation of labelled nucleotides should always be verified by a dot-blot analysis against the appropriate standard dilution series made out from the stock solution of the labelled nucleotides. A tested control sample, made from total genomic DNA or plasmids, is also useful for measuring consistency between labelling reactions (see Appendix 11).

check probe

1.5.3 Blocking DNA The unlabelled blocking DNA is usually from a related species that has much DNA in common with the probe species. The RNase-free total genomic DNA needs to be fragmented to about 100 to 200 bp, for example by autoclaving for 5 min at 15 psi. The amount of blocking DNA is varied, from Ox up to 50x the probe amount. Blocking is generally more effective on chromosomes than on Southern blots, probably because the target DNA originating from different species is physically located on different chromosomes or chromosome segments, whereas on a Southern blot the target DNA is sorted out by fragment lengths, and hence common and speciesspecific sequences are mixed. In the cereal group, the amount of blocking required to distinguish between chromosomes of different genera is 0-15x the probe amount (15x in Fig. 1 E), while between species of the same genus or section, up to 50x the probe amount may be required (e.g. 40x in Fig 16).

preparation of blocking

DNA

amount of blocking DNA required

1.5.4 In Situ Hybridization Procedure 1. RNase treatment of chromosome preparations (optional): Apply 200 pl of RNase solution on each slide, place coverslip and incubate in a humid chamber for 1 h at 37 "C. Remove coverslips and wash the slides twice in 2x SSC in a Coplin jar for 5 min each (if glass coverslip is used, allow the slide to stand in the 2x SSC buffer for few min until the coverslips fall off).

2. Transfer slides from the last 2x SSC buffer to the paraformaldehyde solution in a Coplin jar and incubate for 10 min at room temperature in a fume hood.

3. Rinse the slides briefly in 2x SSC and then 3 min in another 2x SSC wash.

hybridization

A

hazard! use fume hood

4. Dehydrate the slides by sequentially replacing the 2x SSC solution with cold 70% ethanol, then with 90 and 99%, 2 min each. Air dry the slides. Genomic In Situ Hybridizations for Whole Chromosome and Genome Analysis

5. In a microfuge tube, prepare 30 pI of probe hybridization mixture per slide using:

Plus sterile water if needed to make up the volume. Mix the components of the probe mixture well, centrifuge briefly and keep on ice. The hybridization stringency used in genomic in situ hybridization is about 80%. 6. Apply the probe mix onto each slide and place coverslip gently without trapping air bubbles. 7. Place the slides in the preheated humid chamber in the heating block. The program is set to perform combined (probe and target) DNA denaturation for 7-10 min at 80-90 "C and hybridization at 37 "C overnight.

stringency control

The denaturation time depends on the species (e.g. those having a high proportion of heterochromatin like wheat and rye need a higher temperature than barley) and slide quality (higher temperature for older slides or slides prepared from old fixed tissue). The programme then slowly cools the preparation to 37 "C (by cooling to 40 "C in five steps with 10 "C intervals and maintaining the temperature at each step for 1 min), and finally holding at 37 "C for hybridization over 4 to 16 h. Stringency control is very important in genomic probing. This refers to the degree to which reaction conditions favour the disassociation of nucleic acid duplexes and may be enhanced, i.e. by increasing temperature, decreasing salt concentration, and increasing formamide or urea concentration - duplexes with high homology withstand high stringency conditions better than duplexes with low homology. The stringency of hybridization and washing can be estimated, and regulated, according to Meinkoth and Wahl (1984), where stringency ( %) is 100 - (Tm -Ta), Tm is dissociation temperature and Ta is hybridization temperature.

M = ionic strength (mol/l) of sodium ion, in SSC solution G+C = 45.5% in cereal genomes n = number of nucleotides in the probe (500 bp by nick translation) Hybridization stringency is affected by the length of the probe after labelling and length varies depending on factors such as DNase K. ANAMTHAWAT-JONSSON eta1

activity in nick translation reactions or primer concentration in random-primer labelling.

8. Place the slides in 2x SSC, in Coplin jar, to allow the coverslips to fall off and wash them further in 2x SSC for 3 min at 37 "C. 9. Wash in the stringent formamide buffer for 10 min at 37 "C. Agitate regularly. The washing stringency is kept about the same level as that of the hybridization, or slightly higher. 10. Wash in 2x SSC for 5 min at 37 "C, then twice in 4x SSC with Tween, 5 min each, also at 37 "C. 11. Apply 100 pI of 5 % BSA to each slide and incubate for 5 min at room temperature. 12. Tip off the BSA solution and apply 50 PI of anti-digoxigeninFlTC to each slide. Place coverslip. Incubate in a humid chamber for 1 h at 37 "C.

No antibody detection is required when using direct fluorochrome conjugated probes. But when using digoxigenin- or biotin-labelled probes, appropriate detection is required. For digoxigenin-labelled probes, use anti-digoxigenin (FAB fragment) conjugated to fluorescein or rhodamine. For biotin-labelled probes, use avidin or streptavidin conjugated to fluorescein, Texas Red or Cy3. The biotin system can be amplified using anti-avidin.

A

hazard! use fume hood

visualization alternatives

13. Wash in 4x SSC with Tween, in Coplin jar, twice for 5 min each at 37 "C, then bring to room temperature. 14. Apply 100 pl of propidium iodide, or DAPI, onto each slide and after 1 min at room temperature, rinse briefly with distilled water and allow to air dry for about 10 min. 15. Place a drop of antifade onto the chromosome preparation, cover with a glass coverslip and squeeze very hard under filter paper. The mountant should be very thin for optimum resolution. 16. Examine the slides in the epifluorescent microscope, using objective 25x to scan the cells and lOOx to view the fluorescent signal and to make photographs. Fluorescein FlTC and the counterstain propidium iodide are examined under the same wavelength (blue excitation). DAPI is visualized with the UV filter and rhodamine or Texas Red with the green excitation filter. Exposure time for photography depends on signal intensity and film speed: 20 to 45 sec for high-speed films, e.g. 400-1000 ASA. Although colour slide films are widely used, we find colour print films (e.g. Fuji Supercolor HG 400 ASA) have a wider exposure latitude, higher contrast and fine grain, and are easier and cheaper to prepare for publication.

analysis and documentation

photography tips Genomic In Situ Hybridizationsfor Whole Chromosome and Genome Analysis

1.6 Troubleshooting Poor Signal

troubleshooting

control of denaturation

The quality of the chromosome preparations and the labelled probe should be checked along with the steps of the procedure. Optimum denaturation of chromosomal DNA can be difficult to achieve, especially in the combined denaturation method, because the probe DNA may require more agressive denaturation conditions. Therefore the probe mixture can be denatured by boiling for 7 min and cooled on ice, before adding to the preparation and following the denaturation procedure described above. Alternatively, the chromosome preparation can be denatured separately in a formamide solution (70% in 2x SSC) for 2-3 min at 65-72 "C (depending on species and preparation quality). After chromosome denaturation, the slide is dehydrated in the alcohol series. The denatured probe is then applied onto the slide for hybridization.

No Signal Differentiation

blotting experiments

If the probe hybridization signal is good, the blocking is probably insufficient: increase the blocking ratio. If the signal is weak, given that the in situ protocol is optimized, then the different genomes may have too small a proportion of genome- or species-specific DNA: Southern experiments should be set up to determine the level of genome similarity before performing the genomic in situ hybridization. High Background

chromosome preparations

Non-specific hybridization: improve the washing procedure or increase the stringency. The chromosome preparation is not clean: cytoplasm may be removed by increasing the time the material is left in 45 % acetic acid. After making the preparation and RNase treatment, it may be treated in lOmM HCI for 10 min at 37 "C, followed by 10 min to 1 hr in pepsin (40-200 units/ml pepsin) in 10 mM HCI to digest proteins.

Acknowledgements We are most grateful to Drs A.R. and I.J. Leitch and W. Mosgoller for sharing the experience of in situ hybridization with us.

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Genomic In Situ Hybridizations for Whole Chromosome and Genome Analysis

In Situ Hybridization Melody Clark Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 2

Fluorescence In Situ Hybridization: Applications in Gene Mapping and Clinical Diagnostics G. A. EVANS and L. SELLER1

2.1 Introduction Cytogenetic mapping is a valuable approach to the localization of genes to chromosomal bands, for determining the order and approximate spacing between cloned markers in physical maps, and for the evaluation of chromosome structure and organization. Initially the use of radioactive probes and the evaluation of hybridization signals by autoradiography provided a valuable technique for mapping genes (Harper and Saunders, 1981). However, the long time periods necessary for development of autoradiographs, the instability of labeled probes over time, the requirement for statistical analysis to determine map position, and limited resolution due to the use of isotopic signals captured by an emulsion layer, severely limited the use of this technique. In the past few years, the use of non-radioactive fluorescence in situ hybridization (FISH) has largely supplanted these techniques. FISH using repetitive DNA probes allows the identification of human chromosomes in somatic cell hybrids (Pinkel et a/. 1986))the detection of numerical and structural chromosomal abnormalities (Selleri et al. 1991a), and the creation of detailed chromosome maps using cosmid or yeast artificial chromosome probes (Lichter et a/., 1990; Selleri et al. 1991b) hybridized to metaphase or interphase chromosomes (Lichter et a/, 1988). FISH using YAC clones, in particular, is an increasingly important technique for physical genomic mapping at high resolution on chromosomes as well as accessing the integrity of mammalian chromosomal segments cloned in yeast cells (Selleri et a/., 1992) (Fig. 2). Techniques for the reduction of the background hybridization signals of repetitive DNA elements have been introduced (Landegent et al. 1987) allowing detailed analysis and mapping of probes as small as a few hundred base pairs in length. With FISH, essentially the same techniques, with minor variations, can be applied for the analysis of normal chromosomes, using cosmid, YAC or other DNA fragments as probes, and using one or more fluorochromes. The technique utilized can provide a variety of resolutions for different applications. For instance, the hybridization of a cosmid probe labeled with biotinavidin-FITC to banded normal human chromosomes allows a gene

metaphase chromosomes

M. Clark (Ed.), In Situ Hybridization. 0 Chapman & Hall, Weinheim. ISBN 3-8261-0038-7

multi-probe FISH

interphase FISH

to be localized to a specific chromosome and allows assignment of a cytogenetic position to a resolution defined by the band. It is usu.al to refer to the position of a gene localized in this way by the band location, i.e. 11q12-13. The approximate resolution given by this application of FISH is in the range to 10 to 30 Mb. The same technique analyzed by confocal microscopy where the results are determined as percent of chromosome length, or Flpter, usually give resolutions in the range of 2 to 10 Mb (Lichter et al, 1990). Multi-probe FISH can allow the more detailed simultaneous mapping of genes to chromosomes and extend the resolution achieved by fluorescence hybridization. For example, in metaphase chromosome analysis, two cosmid probes are detectable as two distinct hybridization signals when they are separated by at least 1 to 2Mb (Giovannini et al. 1992).When the probes are located closer than this distance, the signals usually merge forming a single hybridization spot of a mixed color. An FlTC (green-yellow) probe co-hybridized with at Texas-Red (red) probe gives an orange hybridization signal when less than 1Mb apart and separate green-yellow and red dots when separated by more than 2Mb. Essentially the same hybridization technique carried out using interphase nuclei rather than metaphase chromosomes can give a resolution down to 40 kb. Two separate hybridization signals are generally seen at greater distances and a single merged spot at smaller distances (Giovannini et al, 1992).

Figure l A , B. Single color FISH. FISH analysis using a single biotin-labeled cqsmid clone containing the CTNF (ciliary neurotrophic factor) gene mapped to chromosome llq12. The probe is located by fractional chromosomal length (Flpter) and the vertical bar indicates the range of Flpter values obtained on measuring 30 individual chromosomes. Reprinted from Giovannini et al. (1993) G.A. EVANS and L. SELLER1

Figure 2A-C. FISH using YAC clones. FISH using YAC clones hybridized to normal human chromosomes to confirm YAC integrity. YAC DNA was labeled with biotin-lldUTP and biotin-11-dCTP and detected using FITCavidin. A Hybridization of a 400 kb YAC to normal metaphases demonstrating hybridization to both chromosome 11 at q24. B Hybridization of a 280 kb YAC demonstrating mapping to human chromosome 1 at q2. C Hybridization of a 250 kb YAC demonstrating hybridization signals on two chromosomes 192 and to a chromosome of the C group. Hybridization signals on two or more different human chromosomes is indicative of a chimeric clone. Reprinted from Selleri et al (1992)

Fluorescence In Situ Hybridization: Applications in Gene Mapping and Clinical Diagnostics

DIRVISH

lnterphase FISH is also extremely useful for the diagnostic analysis of chromosomes in regions where a chromosome anomaly is suspected. lnterphase FISH is capable of giving a yes-no answer to a diagnostic question about the presence or absence of a specific translocation. For instance, a t(ll;22) in the FLI-1 gene of chromosome 11, pathognomonic of Ewing’s sarcoma or peripheral neuroepithelioma, is seen as one pair of hybridization signals and a merged signal (Selleri, 1994). In a separate assay, a interruption of the erg oncogene on chromosome 21 is seen as a merged hybridization signal (Giovannini, 1994). Additional resolution to the limit of about 5 kb can be achieved using essentially the same techniques and similar cosmid probes using distended chromatin rather than metaphase or interphase chromosomes. This technique (DIRVISH) is extremely useful for determining the order of closely spaced probes and the distances separating them (Parra and Windel, 1993). It also has the potential for wide use for determining the coverage of YAC or cosmid clone contigs spanning regions of interest and allows precise measurement of the gap distances separating cloned areas. This technique is likely to find wide applications in the high resolution physical mapping efforts necessary to produce the templates for sequencing the human genome. So, FISH has a variety of applications in gene mapping and diagnostics, using a variety of different probe types. This chapter will outline the techniques described above for FISH analysis applicable to the mapping of cloned genes to chromosomal locations, physical genomic mapping and diagnostic applications using single copy probes labeled with one or more fluorochromes.

2.2 Materials and Chemicals 2.2.1 Buffers and Reagents Cell lines: Human cell lines carrying a normal complement of chromosomes are most useful for in situ hybridization mapping. Human fibroblast cell line CRL 1634, obtained from the Human Genetic Mutant Cell Repository (Cambden, N.J.) has been shown to have a normal male karyotype and is suitable for most mapping purposes. Before use, any cell line should be characterized by detailed cytogenetic analysis to confirm a normal human karyotype. Collagenase I: 2mg/ml in PBS Hybridization buffer: 50% formamide, 2X SSC, pH 7.0, 10% dextran sulfate Lysis solution: 0.45 M EDTA, 10 mM TRIS, pH 8.0, 1 % sodium N-lauroyl sarcosinate, 0.5 mg/ml proteinase K Paraformaldehyde: 4 % in PBS with 50mM MgCI2

PBS: 130mM NaCV7mM Na2HP04/3mMNaH2P04pH 7.0

G.A. E V A N S and 1. SELLER1

Figure 3A-E. DIRVISH. Analysis of a 300 kb region of chromosome 11q24 encompassed by YAC ySL-B45Cll using individual cosmid clones by DIRVISH. AThe bulk of 1000-5000 individual cells from a normal fibroblast cell line, resuspended in one droplet of phosphate buffered saline and lysed in situ on the slide, can be seen at one end of the slide. The DNA stream, running down the slide after it has been tilted to a vertical position, is visible after DAPl staining. B DAPl stained chromatin fibers extending down the slide, which is shown here horizontally. C Enlargementof a digoxigenin-labeled cosmid detected with rhodamine (red) on a chromatin fiber. D A digoxigenin-labeled cosmid, detected with rhodamine (red), and biotinlabeled cosmid, detected with fluorescein (green), were co-hybridized. E A digoxigenin-labeled cosmid, detected with rhodamine (red), a biotin-labeled cosmid, detected with fluorescein (green), and a second biotin-labeled cosmid, also detected with fluorescein (green),were co-hybridized in this experiment.The order of the hybridization signals can be determined as cosmid 1 (red)-gap - cosmid 2 (green) - gap - cosmid 3 (green) . Three red dots of rhodamine signal, present between cosmid 1 (red) and cosmid 2 (green), were interpreted as background; this interpretation is consistent with the gap size observed in other DIRVISH preparations (D)and with the gap size determined from physical mapping of this regions. Reprinted from Selleri et al. (1995)

FluorescenceIn Situ Hybridization: Applications in Gene Mapping and Clinical Diagnostics

PN buffer: 01 M phosphate buffer, pH 8.0,O.l % Nonidet P-40. Pre-antibody block: 4 % goat serum/0.5 % Nonidet P-40/0.1M Na2HP04pH 8.0 Proteinase K: 0.5pg/ml in 20mM Tris/2mM CaCI2 RNase A solution: 100pg/ml in 0.3M NaCV30mM sodium citrate

SCE buffer: 1 M sorbitol, 0.1M trisodium citrate, pH 5.8, 10 mM EDTA SSC buffer (1X): 0.15 M NaCI, 0.015 sodium citrate TBE buffer (1X): 90 mM TRlS base, 90 mM boric acid, 2.5 mM EDTA

2.2.2 Chemicals Anti-DIG Fab fragment Boehringer Mannheim antifade solution ONCOR Biotin-11-dUTP, Biotin-11-dCTP Boehringer Mannheim Vector Labs biotinylated antiavidin DAPl Sigma Boehringer Mannheim deoxyribonucleotide triphosphates DIG-11-dUTP DNase I

Boehringer Mannheim Boehringer Mannheim

DNase-free RNase Dulbecco’s modified Eagle’s medium (DMEM) fetal calf serum (FCS) FlTC conjugated avidin FITC-dUTP, FITC-dCTP formamide goat serum human Cot-1fraction DNA human placental DNA lyticase oligodeoxyribonucleotide primers (random hexamers) paraformaldehyde propidium iodide proteinase K Sheep antidigoxigenin Texas Red avidin Texas Red Sheep IgG (H+L) [Donkey anti sheep] trypsin

Boehringer Mannheim Cellgro from Fischer

1 214 667 S1370-5 1093 070 BA0300 D9542 nick translation kit: 976 776 109 3088 nick translation kit: 976 776 1119915 15-013-LM

Geminii 100-106 Vector Labs A2001 Boehringer Mannheim 1373 242 International Biotechnolgies, Inc. I872024 Gibco/BRL 16210-015 15279-011 Gibco/BRL D7011 Sigma L5263 Sigma 18247-015 Gibco/BRL Sigma Sigma Boehringer Mannheim Boehringer Mannheim Boehringer Mannheim Boehringer Mannheim

P6148 P4170 745-723 1214 667 100 215 713-076-147

Cellgro from Fischer

25-052-Ll

2.2.3 Equipment Standard, readily available, laboratory equipment is used throughout these protocols. Most of the microscopy involves a conventional epifluorescence microscope, where more specialised equipment is involved, this is detailed under the microscopy sections: 2.6.5 and

2.7.4. G.A. EVANS and L. SELLER1

2.3 FISH Using Single Copy Cosmid Probes Single copy hybridization for chromosomal mapping utilizes metaphase chromosome spreads prepared from cytogenetically normal human cells or other mammalian cell lines. The chromosomes are spread on microscope slides and may be banded before hybridization. A unique copy probe, such as a cosmid or plasmid, is labeled and hybridized to the metaphase spread under conditions designed to minimize or suppress the signals of repetitive DNA elements such as Alu, Kpn 1, LINE or other repeats. This usually involves a pre-hybridization step where the probe or chromosome spread, or both, are hybridized to saturation with a nonlabeled probe containing a high concentration of repetitive elements. The hybridization signal is then detected by fluorescence microscopy. A typical result is shown in Fig. 1.

2.3.1 Preparation of Metaphase Chromosomes NB: It is essential to confirm the normal karyotype by standard cytogenetics before using cell lines for FISH studies. Metaphase chromosome spreads are generally prepared as follows:

1. Cells are grown in standard media in tissue culture plates and growth synchronized. Colcemid at a concentration of 0.1 pg/ml is added to a growing cell culture and the cells incubated at 37 "C for 30 to 60 minutes.

check karYoVPe

me+aphasechromosomes

2. The cells are then collected by centrifugation at 500g for 10 minutes and suspended in ice cold 0.075 M KCI for 13 to 18 minutes.

3. Chromosome spreads are prepared by dropping cells onto microscope slides from a distance of 3 to 18 cm from a glass Pasteur pipette. The cell droplets are dried, fixed by emersion in methanol/glacial acetic acid (3:1) and the emersion repeated for 6 to 8 washes of 30 seconds each. The slides are then air dried and stored at -20 "C until needed for FISH.

2.3.2 Preparation of Metaphase Chromosome Slides for Hybridization Metaphase chromosome spreads, prepared as described above, are treated with RNase A solution for 1 hour at 37 "C, followed by dehydration in a series of 70%, 85% and 100% ethanol baths. Proteinase K digestion is then carried out at a concentration of 0.5pg/ml in 20 mM Tris/2mM CaCI2 at 37 "C for 7 minutes. The slides are fixed in 4 % paraformaldehyde in PBS with 50 m M MgCl, at room temperature for 10 minutes. Fluorescence In Situ Hybridization: Applications in Gene Mapping and Clinical Diagnostics

2.3.3 Preparation and Labeling of Probes Cosmid DNA is generally prepared for use as a FISH probe by a cosmid purification procedure using alkaline lysis of cells, extraction of the cosmid DNA from the cell lysate using phenokhloroform extraction, followed by purification by ethanol precipitation (Birmboim and Doly, 1979).The cosmid DNA is resuspended at a concentration of 1 pg/pI in TE buffer and usually is of sufficient purity for immediate use as a FISH probe. A greater degree of purity can be achieved by CsCl density centrifugation and banding (Sambrook et al, 1989). To prepare cosmid DNA as a FISH probe, 50 ng to l p g of cosmid DNA is labeled using random hexamer primed D N A synthesis (Feinberg and Vogelstein, 1983) and using biotinylated dCTP as a label (see Appendix I). The probe is then used directly for hybridization reactions without further purification. Following labeling, the fluorescently labeled probe is stored at 4 "C prior to use and is generally useful for about two weeks after labeling.

2.3.4 Hybridization of Cosmid Probes 1. The labeled DNA is prepared for hybridization to human metaphase chromosome spreads: 25 to 1OOpg of biotinylated cosmid DNA is precipitated with 21.19 of human placental DNA or 1-2 pg of human Cot-1 DNA. The carrier DNA has previously been sonicated to a final size range of 200 to 400 bp. 2. The biotinylated cosmid probe and carrier DNA is resuspended in lop1 of hybridization buffer and denatured at 75 "C for 30 minutes. Preannealing of repetitive sequences is carried out for 30 minutes at 42 "C before application of the hybridization mixture to the denatured slides.

3. Hybridization is carried out for 16 to 24 hours at 37 "C in a humidif ied chamber. 4. Following hybridization, the slides are washed three times for 3 minutes in 4X SSC containing 0.1 YOTween 20 at 37 "C. A final wash is carried out at 65 "C in 0.1X SSC for 20 minutes. 5. The slides are then treated with 2 alternating layers of FlTC conjugated avidin and biotinylated goat antiavidin as follows: FlTC conjugated avidin at 5$/ml in 1 % BSA, 4X SSC, 0.1 YOTween 20 is placed on the slide and incubated for 3 minutes at room temperature. The slide is washed in in 4X SSC, 4X SSUO.1 % Triton X100, and PN buffer, respectively, for 3 minutes. Biotinylated goat antiavidin at 5pl/mI in 1 % BSA, 4X SSC, 0.1 % Tween 20 is placed on the slide and incubated for 3 minutes at G.A. EVANS and L. SELLER1

room temperature, followed by washing in 4X SSC, 4X SSC/

0.1 % Triton X100, and PN buffer for 3 minutes. 6. The treatment with FlTC conjugated avidin is repeated followed by washing in 4X SSC, 4X SSUO.1 % Triton X100, and PN buffer. 7. The treatment with biotinylated goat antiavidin is then repeated as above followed by washes in 4X SSC, 4X SSC/O.l % Triton X100, and PN buffer. 8. After the final wash, the slides are mounted in antifading sohtion containing 2OOpg/ml propidium iodide counterstain, and evaluated on a conventional epifluorescence microscope.

2.4 Multicolor FISH Using Cosmid Probes FISH using multiple probes is carried out essentially as for single copy, utilizing two different probes labeled with two different fluorochromes. A wide variety of fluorochromes are now available as well as several “sandwich” techniques for fluorescent tagging. In addition, the availability of deoxyribonuleotide triphosphates directly labelled with fluorochromes allows a simpler and more widely applicable labelling technique to be applied to many experimental conditions (Ballard and Ward, 1993).The following is a conventional dualcolor labelling protocol using two “sandwich”-type labeling protocols. In this protocol one probe Typically, one probe is labeled with FITC-dUTP and a secondis labeled with biotinylated-11-dUTP and detected by treatment with Texas Red avidin. The second probe is labeled, under essentially identical conditions, using digoxigenin-lldUTP and detected using FlTC labeled antidigoxigenin. Alternate procedures using FITC-11-dUTP are possible by substituting components of the labeling reactions.

2.4.1 labeling of Probes and Hybridization Reactions For multicolor FISH detection, two DNA probes, cosmid or YACs, are purified as described in other procedures in this chapter then labeled individually, One probe is labeled using a random hexamer primed synthesis reaction as described in the previous protocol. The second probe is similarly labeled using an identical reaction, but substituting 11-digoxigenin dUTP instead of 11-biotin dUTF?

1. Following labeling of the probes and preparation of interphase or metaphase slides for hybridization, the hybridization reaction is set-up as follows: 25 to lOOpg of the biotinylated cosmid DNA is mixed with 20 to lOOyg of the dioxigenin-labeled DNA and 2pg of human placental DNA or l-2yg of human Cot-1 DNA (sonicated to a final size range of 200 to 400 bp). The probes

multicolor FISH

FluorescenceIn Situ Hybridization: Applications in Gene Mapping and Clinical Diagnostics

are ethanol precipitated by adding 0.1 volume of 4N sodium acetate, pH 4.5, and 3 volumes of cold 95% ethanol. The sohtion is frozen on dry ice for 5 minutes, then thawed between fingers and the precipitate collected by centrifugation at 10,000 rpm in a microcentrifuge.

2. The probekarrier DNA mixture is resuspended in lop1 of hybridization buffer and denatured at 75 "C for 30 minutes. Preannealing is then carried out by cooling to 42 "C and incubation for 30 minutes. The hybridization mixture is then applied to the denatured slides and hybridization is carried out for 16 to 24 hours at 37 "C in a humidified chamber.

3. Following hybridization, the slides are washed three times for 3 minutes in 4X SSC containing 0.1 % Tween 20 at 37 "C.

4. A final wash is carried out at 65 "C in 0.1X SSC for 20 minutes. Development of the two potential hybridization signal is carried out as follows:

5. The slides are then treated with Texas Red conjugated avidin at 5pl/ml in 1 % BSA, 4X SSC, 01 % Tween 20 for 3 minutes at room temperature.

6. The slide is then washed in 4X SSC, 4X SSUO.1 % Triton X100, and PN buffer, respectively, for 3 minutes.

7. The treatment with Texas Red conjugated avidin is followed by treatment with FITC-conjugated antidigoxigenin at 5pVml in 1 % BSA, 4X SSC, 0.1 % Tween 20 for 3 minutes at room temperature followed by washing in 4X SSC, 4X SSUO.1 % Triton X100, and PN buffer. If necessary, each treatment may be repeated up to three times to increase fluorescent signal. The number of treatments is usually determined empirically to balance the signal of each DNA probe. optimisation

8. After the final wash, the slides are mounted in antifading and evaluated on a conventional epifluorescence microscope.

2.5 lnterphase FISH Analysis lnterphase FISH allows the detection of hybridization signals of unique single copy DNA fragments as individual points of hybridization in a fixed interphase nucleus. This allows quantification of chromosome number as individual dots, or analysis of chromosome translocation breakpoints where two or more individual DNA segments surrounding the region of potential translocation are used simultaneously. G.A. EVANS and L. SELLER1

2.5.1 Preparation of lnterphase Nuclear Slides from Short Term Cultures Short term cultures can be established directly from fresh tissue obtained at biopsy, or from tumor material.

1. Tumors are minced into small pieces with a sterile razor blade or scalpel and incubated for 1 hour at 37 "C in PBS containing 2 mg/ml Type I collagenase I. Every 10 minutes the solution is vortexed briefly to facilitate the detachment of tumor cells from the stroma and the tissue debris is allowed to settle.

interphase FISH

2. The suspension containing individual cells is then collected and centrifuged at 1000 rpm for 10 minutes. The cell pellet is collected and washed in Dulbecco's modified EagleS medium (DMEM)containing 15 % DMEM supplemented with 15 % FCS.

3. Cultures are incubated at 37 "C in a humidified atmosphere in 5 % C 0 2 . In most cases cells are ready for harvesting within 4 days after the culture is established.

4. Cells are removed from the tissue culture plate by brief trypsin treatment (1 to 3 minutes at 50pg/ml in serum-free DMEM media), washed twice in cold PBS, and treated with a hypotonic solution of 0.075 M KCI for 30 minutes.

5. The suspension is applied to microscope slides by dropping and fixed with three changes of methanoVacetic acid

(3:l).

2.5.2 Direct Preparation of lnterphase Spreads Alternately, interphase nuclear preparations may be prepared directly from fresh tissue.

1. Tissue is minced into small pieces with a sterile razor blade or scalpel and incubated for 1 hour at 37 "C in 2 mg/ml collagenase type I in PBS. Every 10 minutes the solution is vortexed briefly to facilitate detachment of tumor cells from the stroma.

2. The debris is allowed to settle and the cell suspension is then removed and centrifuged at 1000 rpm for 10 minutes. The cells

are resuspended in PBS, washed twice in cold PBS, then resuspended in 0.075M KCI and incubated on ice for 30 minutes.

3. Nuclei are applied to a microscope slide and fixed using three changes of methanoVacetic acid.

2.5.3 FISH Biotin labeling, suppression of repetitive signals hybridization and analysis are carried out essentially as in the previous procedure. To aid in the detection of weak hybridization signals, labeling can be carFluorescence In Situ Hybridization: Applications in Gene Mapping and Clinical Diagnostics

ried out using a random primer extension reaction where dTTP is replaced with bio-11-dUTP and dCTP is replaced with bio-11-dCTP at final concentration of 20yM. This effectively increases biotin incorporation during labeling.

2.6 FISH Analysis Using YAC Clones 2.6.1 Preparation of Metaphase Chromosomes Metaphase chromosome spreads are generally prepared as follows:

FISH using YAC clones

1. A synchronized culture of cells with an apparent normal karyotype is prepared. Colcemid is added to the culture to a concentration of 0.1yg/ml and the cells are incubated at 37 "C for 30 to 60 minutes.

2. The cells are then collected by centrifugation at 5 0 0 x g for 10 minutes and suspended in ice cold 0.075 M KCI for 13 to 18 minutes.

3. Standard chromosome spreads are prepared by dropping cells onto microscope slides and the cells are fixed using methanol/ glacial acetic acids (3:l) for 6 to 8 washes of 30 seconds each. The slides are dried and stored at -20 "C until needed for FISH.

2.6.2 Isolation of YAC DNA YAC clone DNA is isolated from yeast cultures by preparative pulsed field gel electrophoresis.

1. Yeast cells are collected by centrifugation at 3000 x g for 20 minutes and the cell pellet washed twice in 50mM EDTA, pH 8.0.

2. The pellet is then resuspended in 0.4 ml SCE buffer containing 100 mM 8-mercaptoethanol and 1000 U/ml lyticase. The suspension is then mixed with 0.4 ml of 1 % low-melting point agarose in SCE buffer and poured in 80yl slots in a Plexiglas PFG plug mold.

3. The plugs are allowed to solidify at 4 "C and then incubated in SCE buffer containing 100 m M 8-mercaptoethanol for 5 to 12 hours at 37 "C.

4. The buffer is replaced with lysis solution and incubated in DNAase-free Rnase A (2.5 U/ml) at 37 "C for 1 hour. The plugs are then stored in 0.5M EDTA at 4 "C until needed. 5. YAC DNA embedded in agarose plugs is equilibrated in 0.5X TBE buffer and embedded in the slots of a 1 % low melting point agarose gel. G.A. EVANS and L. SELLER1

6. Pulsed field gel electrophoresis is carried out in 0.5X TBE buffer at 16 "C for 24 hours at 10V/cm with ramping times from 10 to 55 seconds. These ramping times are appropriate for YAC clones in the range of 100 to 600 kb and may be modified for larger clones. Concatomerized lambda DNA or S. cerevisiae chromosomes are utilized as size markers.

optimisation

7. The gel is stained with ethidium bromide at 10pg/ml, the band containing the YAC DNA visualized by UV transillumination and is excised with a razor blade. The blocks are stored at 4 "C until used.

2.6.3 Biotin Labeling of YACs 1. A YAC DNA plug excised from a preparative PFG gel is heated at 65 "C and diluted with 5 volumes of TBE buffer. Prior to labeling, 25 to 50pI of the melted agarose containing YAC DNA is treated for 1 hour at 14 "C with DNAase I. The conditions of treatment varied with the characteristics of the

YAC and particular preparation should be determined empirically.The optimal final concentration of DNAse I is usually in the range of 500 top 1 :2000 of a 1 mg/ml stock solution.

optimisation of DNAse treatment

2. Labeling is then performed using a random hexamer primed reaction (Feinbergand Vogelstein, 1983)where dTTP is replaced with bio-11-dUTPat 20pm final concentration. Following labeling the biotinylated YAC probe is purified from unincorporated nucleotides by passing the reaction mixture through Sepadex G50 in 1 ml spun columns.

2.6.4 FISH 1. Metaphase chromosome spreads, prepared as in the previous method, are treated with RNase A solution for 1 hour at 37 "C, followed by dehydration in a series of 70%, 85% and 100% ethanol baths.

2. Proteinase K digestion is then carried out at at 37 "C for 7 minutes. The slides are fixed in 4 % paraformaldehyde in PBS with 50 mM MgCI2at room temperature for 10 minutes. 3. Hybridizations are carried out as follows: 25 to 50 ng of biotinyluted YAC DNA is precipitated with 2pg of human placental DNA or 1-2pg of human Cot-1 DNA. The carrier DNA has previously been sonicated to a final size range of 200 to 400 bp. The biotinylated YAC probe and carrier DNA is resuspended in lop1 of hybridization buffer and denatured at 75 "C for 30 minutes. Fluorescence In Situ Hybridization:Applications in Gene Mapping ond Clinical Diagnostics

4. Preannealing of repetitive sequences is carried out for 30 minutes at 42 "C before application of the hybridization mixture to the denatured slides.

5. Hybridization is carried out for 16 to 24 hours at 37 "C in a humidified chamber.

6. Following hybridization, the slides are washed three times for 3 minutes in 4X SSC containing 0.1 % Tween 20 at 37 "C. A final wash is carried out at 65 "C in 0.1X SSC for 20 minutes.

7. The slides are then treated with 2 alternating layers of FITC conjugated avidin and biotinylated goat antiavidin both at 5pl/ml in 1 % BSA, 4X SSC, 0.1 % Tween 20 (as described in 2.3.4). After the final wash, the slides are mounted in antifading solution containing 200pg/ml propidium iodide counterstain, and evaluated on a conventional epifluorescence microscope. A typical result is shown in Fig. 2.

2.6.5 Microscopy

microscopy (confocal) and dOCU~entation

Higher quality images can be obtained by confocal microscopy using a BioRad MRC 600 laser scanning confocal microscope. Narrow band pass filters are used to obtain separate images of each fluorochrome (550 nm filter for FITC; 610 filter for propidium iodide). The two separate images are stored and overlaid electronically with digital filtering to remove background and optimize visualization of the hybridization signal. The fluorescent signals are quantitated directly, signal to noise ratio enhanced by Kalman optical filtering, and images stored digitally. The map position of a single hybridization signal can be determined and quantitated as the FL ( fractional length) relative to a fixed reference point, such as the short arm telomere, pter (Lichter et al. 1990).Hybridization signal position should be determined routinely from an average of 15 to 30 measurements and expressed as FLpter +/- standard deviation. Greater than 90 % of metaphases should show hybridization signals on both sister chromatids of the metaphase chromosomes.

2.7 Direct Visual In Situ Hybridization (DIRVISH) DIRVISH allows the highly detailed analysis of chromatin through the hybridization of unique single copy cosmids to extended or "stretched" chromatin preps. DNA from isolated cell lines is stretched using modifications of the protocol of Parra and Windle, 1993.

G.A. EVANS and L. SELLER1

2.7.1 Preparation of Stretched DNA 1. 1 to 5 x lo6 cells/ml from a karyotypically normal human fibroblast cell line are collected by centrifugation at lOOOg and resuspended at a concentration of 1000 to 5000 cells/yl in PBS.

2. A 2pl droplet of this cell suspension is placed on a microscope

DlRVlSH

slide and air dried at room temperature.

3. The cells are lysed by applying 5ml of 0.5% SDS, 50 mM EDTA, 200 mM Tris (pH 7.4) over the surface of the dried cell droplet. After 5 minutes of incubation, the slide is tilted to a vertical position allowing a stream of DNA to run down the slide (Fig. 3 A, B).

4. Slides are then air dried and chromatin fibers fixed with methanoVacetic acid (3:l) for 5-10 min. When dry, the slides are stored at -20 "C in boxes containing Silica gel.

2.7.2 FISH Hybridization 50-100 ng of a cosmid probe, or other appropriate probe, are labeled by random priming synthesis with a mixture of biotin-dUTP and biotin-dCTP The reactions are carried out essentially as described in the previous protocols substituting biotinylated nucleotides for the non-labeled trinucleotides. A second cosmid is labeled using digoxigenin-dUTP also essentially as described in the previous protocols. 25-100ng of biotin-labeled probe, 25-100ng of the digoxigenin-labeled probe and 2yg of human Cot-1DNA are mixed, incubated for 30 minutes at 42 "C and ethanol precipitated as described above. Hybridization to fixed DNA on the slides is then carried out essentially as described for metaphase FISH (2.3.4). Following hybridization, slides are washed in 50 % formamide/2X SSC at 45 "C for 10 minutes, followed by 2X SSC at 45 "C for 10 minutes and finally in 0.1X SSC at 60 "C for 10 minutes.

2.7.3 Detection and Analysis The detection of biotin-labeled cosmid DNA is carried out first, essentially as described above for multicolor fish (2.4.1).The detection is carried out by sucessively treating the slides with fluorochromeconjugated avidin followed by anti-avidin antibody followed again by by fluorochrome avidin. The detection of digoxigenin labeled cosmids is performed using modifications of the protocol of Parra and Windle (1993) and Selleri et al. (1995).

NB: All antibodies are diluted in "pre-antibody block" solution (4 % goat serum/0.5 % Nonidet P-40/0.1 M Na2P04at pH 8.0) 1. Slides are incubated in pre-antibody block solution for 20 minutes at 37 "C. Fluorescence In Situ Hybridization: Applications in Gene Mapping and Clinical Diagnostics

2. The slides are then incubated with mouse anti-digoxigenin antibody at a final concentration of 10 mg/ml for 25 minutes at 37 "C.

3. The slides are washed at room temperature in 4xSSC, 4xSSC/ 0.1 % triton X100, and PN buffer, respectively for 3 minutes each. 4. The pre-antibody block is applied to the slides again for 20 minutes at 37 "C, followed by incubation with goat anti-mouse Ig-F(ab') fragment at a final concentration of 10 mg/ml, for 25 minutes at 37 "C. 5. The slides are washed again in 4xSSC, 4xSSC/O.1 % triton X100, and PN buffer, respectively for 3 minutes each at room temperature.

6. The slides are again incubated with the pre-antibody block solution for 20 minutes at 37 "C, and then with Rhodamineconjugated anti-digoxigenin Fab fragment at a final concentration of 25 mg/ml, for 30 minutes at 37 "C (alternate fluorochromes may be used as needed).

7. After the final washings again in 4xSSC, 4xSSC/O.l % triton X100, and PN buffer, respectively for 3 minutes each, an anti-fade solution containing DAPI stain is applied to the slides.

2.7.4 Microscopy

microscopy and documentation

Fluorescence microscopy is carried out using a Zeiss Axiophot Fluorescence microscope with a XlOO Plan Neofluar objective lens and a triple band-pass filter (Omega), which allows the simultaneous visualization of DAPI, Fluorescein and Rhodamine, avoiding the image shift that occurs when filters are switched. Photography in epifluorescence mode is performed using Ektachrome ASA P1600 slide film (Kodak), using a Zeiss microscopic camera, with exposures from 30 to 120 seconds. Slides are then scanned using a Microtek scanner to obtain digital images. Colors were enhanced with Adobe Photoshop and Canvas 3.0 on a Macintosh Quadra 840 computer. For typical results see Fig. 3.

2.8 Troubleshooting

troubleshooting

G.A. EVANS and L. SELLER1

The techniques for FISH analysis, using single or multiple labels are necessarily simple. Nevertheless, each step and component is critical to the success of the experiment and problems with the techniques can usually be traced to one of several factors. Most of the common problems with FISH technique will relate to either:

Lack of a sufficient fluorescent signal: Poor slide preparation resulting in inadequately denatured chromosomal DNA. Insufficiently labeled probe Excessive background (fluorescence background too great) Excessive competitive hybridization (preannealing)

High background: Inadequate preparation of slides resulting in a high nonspecific adherence of labeled material Impure or improperly prepared reagents Low quality fluorescent label Insufficient prea nnealing.

Inadequate Slide Preparation High quality slide preparation is essential to insure high quality FISH results. While the procedures for slide preparation are simple, there is much art in the process of good slide preparation and adequate denaturation to allow high quality FISH results: Preparation technique: Metaphase slide preparation using the droplet technique as described here requires some practice and, in the case of initially inadequate results, instruction from someone experienced in this technique can be useful. Since the technique is essentially the same as that used in standard karyotype analysis, clinical genetics laboratories frequently are able to be of assistance to the inexperienced molecular geneticist. Insufficientdenaturation:

A potential cause of inadequate hybridization signal or high background can be the insufficient denaturation of chromosomal DNA on the slide. In the case of this suspected problem, increasing the concentration of paraformaldehyde from 4 % to 6 % may result in a stronger signal. If an aged solution is used, often replacing the paraformaldehyde with a fresh solution may solve the problem. Care should be taken, however, since excessive paraformaldehyde concentration can lead to high background signal. Alternatively, treatment with formamide (70% in 2X SSC, pH 7.0, at 70 degrees for 3 minutes) may be substituted for paraformaldehyde fixation in the case of high background.

Insufficient labeling of Probe DNA Adequate labeling of probe: By far the most common problem relates to the insufficient fluorescent signal associated with a DNA probe, either by virtue of in adequate labeling by random-hexamer primed synthesis. Appropriate labeling Fluorescence In Situ Hybridization:Applications in Gene Mapping and Clinical Diagnostics

with alternate radioactive labels in place of fluorescent ones can control for this problem as well as assuring the purity and freshness of all components of the reaction. Inadequate labeling may also be attributed to impurities in the DNA probe preparation. While adequate results can very frequently be obtained using mini-prep or quick prep procedures, should FISH with a particular probe fail, repeating the procedure with a more carefully prepared DNA probe is often the most direct route to solving the problem. Also see troubleshooting in Appendix I. Inadequate development with a second, sandwich reagent: In the case of sandwich procedures, if used, repeating the development and washing cycle additional times can sometimes make up for low specific activity of the probe.

Inadequate or Excessive Competitive Hybridization Hybridization in the context of FISH requires a balance between the use of large DNA probes to insure adequate fluorescent signal, and the preannealing to non-labeled repetitive DNA to remove the nonspecific hybridization to repeats. Repetitive sequences represent technique problems which may be clone-specific depending upon the density of repeats present. In general, however, the extent of “blocking” hybridization to remove non-specific background should be empirically determined by varying the time of hybridization and the concentration of repetitive sequences used. Over hybridization/blocking: This will result in diminution of the specific signal, resulting in a faint hybridization and the requirement for greater development procedures or instrumentation of higher inherent fluorescence sensitivity. This is because extensive preannealing results in elimination of some of the low copy, as well as high copy repeat signals. The problem may be worse using total human placental DNA, which contains the unique sequence DNA, instead of purified repetitive DNA. The probe may be overcome by using Cot-1 DNA instead of total human placental DNA or by decreasing the hybridization time or the concentration of non-labeled repeats in the preannealing reaction. Inadequate hybridization/blocking: In many cases, excessive “background” or non-specific hybridization may be the result of inadequate blocking hybridization allowing some of the repetitive elements in the probe to cross-hybridize. In this circumstance, the solution is to increase the concentration of repetitive blocking reagents (human placental DNA or Cot-1 DNA) until the background disappears leaving the specific signal. In both cases, the use of appropriate controls will aid immeasurably in the ability to titrate the specific versus non-specific hybridization signals. G.A. EVANS and L. SELLER1

low Fluorescent Sensitivity of Instrumentation

FISH technology is also a balance between the extent of probe labeling and development, and the inherent sensitivity of the available instrumentation.While quite acceptable FISH results may be obtained using conventional epifluorescence microscopy, in general, confocal microscopy or low-light CCD cameras allow more sensitivity and thus a greater latitude in the types of probes which may be used. Conventional microscopy is generally inadequate for detection of hybridization of probes less than cosmid-size, about 40 kb. It is potentially possible, and has been demonstrated, that the use of more sophisticated digital instrumentation will allow detection of hybridization signals of 1 to 5 kb probes labeled with a single color. In addition, the use of confocal or CCD-based microscopy allows a wide variety of digital image enhancing procedures which can be quite effectively applied to remove or reduce background or enhance weak hybridization signals (Ballard and Ward, 1993). These techniques must be used soberly, however, to avoid enhancing faint hybridization signals to the point of generating data of questionable significance. References Ballard, S.G.; Ward, D.C., (1993) Fluorescent in situ hybridization using digital imaging microscopy. J. Histochem. Cytochem. 41, 1755-1759 Birmboim, H.C; Doly, J., (1979)A rapid alkaline extraction procedure for screening recombinant DNA. Nucl. Acids. Res. 7, 1513-1523. Feinberg, A,, Vogelstein, B., (1983)A technique for radiolabelling restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. Giovannini, M., Selleri, L., Biegel, J., Scotlandi, K., Emanuel, B.S., Evans, G.A., (1992)lnterpahse cytogenetics for the detection of the t(ll:22)(q24;q12)in small round tumors. J. Clin. Invest. 90: 1911-1918. Giovannini, M., Romo, A.J., Evans, G.A., (1993)Chromosomal localization to the human ciliary neurotrphic factor gene (CNTF) to llq12 by fluorescence in situ hybridization. Cytogen. Cell. Genet. 63:62-63. Giovannini, M., Biegel, J.A, Serra, M, Wang, J.Y, Wei, Y.H, Nycum, L, Emanuel, B.S, Evans, G.A. , (1994) EWS-erg and EWS-Flil fusion transcripts in Ewing’s sarcoma and primitive neuroectodermal tumors with variant translocations. J. Clin. Inves.94:489-496 Harper, M.E., and Saunders, G.F., (1981)Localisation of single copy DNA sequences on G-banded human metaphase chromosomes by in situ hybridization. Chromosoma 83:431-439. Landegent, J.E., Jansen in de wal, N., Dirks, R.W., Baas, F., van der Ploeg, M., (1987) Use of whole cosmid cloned genomic sequences for chromosomal localisation by non-radioactive in situ hybridization. Hum. Genet. 77: 366-370 Lichter, I?, Chang Tang, C.J., Call, K., Hermanson, G, Evans, G.A., Housman, D., Ward, D., (1990) High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247:64-69 Lichter, i?, Cremer, T., Borden, J., Manuelidis, L., Ward, D.C., (1988)Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Hum Genet. 80:

224-234 Parra, I., Windle B., (1993)High resolution visual mapping of stretched DNA by fluorescent hybridization. Nat. Genet. 5: 17-21 Fluorescence In Situ Hybridization:Applications in Gene Mapping and Clinical Diagnostics

Pinkel, D., Straume, T., Gray, J.W., (1986) Cytogenetic analysis using quantitative high sensitivity fluorescence hybridization. Proc. Natl. Acad. Sci. USA 83: 2934-2938. Sambrook, J; Fritsch, E.F; Maniatis, T (1989) Molecular cloning : A laboratory manual. Pub. Cold Spring Harbor Laboratory Press, NY Selleri, L., Hermansan G.G., Eubanks, J.H. Lewis, K., Evans, G.A. (1991~) Molecular localisation of the t(11;22)(q24;q12) translocation of Ewing sarcoma by chromosomal in situ suppression hybridization. Proc. Natl. Acad. Sci. USA 88: 887-891 Selleri, L., Hermanson, G.G., Eubanks, J.H. Evans, G.A., (1991b) Chromosomal in situ hybridization using yeast artificial chromosomes. Genet. Anal. Tech. Appl. 8:59-66 Selleri, L., Eubanks, J.H., Giovannini, M., Hermanson, G.G., Romo, A,, Diabali, M., Maurer, S., McElligott, D.L., Smith, M.W., Evans, G.A., (1992) Detection and characterisation of ,,chimeric” yeast artificial chromosome clones by fluorescent in situ suppression hybridization. Genomics 14536-541 Selleri, L., Giovannini, M., Romo, A,, Zucman, J., Delattre, O., Thomas, G., Evans, G.A.,(1994) Cloning of the entire F L l l gene disrupted by the Ewing’s sarcoma translocation breakpoint on 11924, in a yeast artificial chromosome. Cytogen. Cell Genet. 67:129-136 Selleri, L., Smith, M. W., Holmsen, A.L., Romo, A.J., Thomas, S.D., Paternotte, C., Romberg, L.C.R., Wei, Y.H., Evans, G.A., (1995) High resolution physical mapping of a 250Kb region of human chromosome 11q24 by genomic sequence sampling. Genomics 26:489-501.

G.A. EVANS and L. SELLER1

In Situ Hybridization Melody Clark Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 3

Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ labelling (PRINS): Two Alternatives to Traditional In Situ Hybridization Methods J. HINDKJAR, C. TERKELSEN, S. K0LVRAA, J. KOCH, and L. BOLUND

3.1 Introduction The Primed in situ labelling (PRINS) technique (Koch et al., 1989) is a fast and sensitive alternative to the traditional fluorescent in situ hybridization (FISH). The principle of PRINS (illustrated for DNA in figure 1) is that an unlabelled nucleic acid probe is hybridized in situ to its target sequence which is subsequently labelled. The labelling is performed via a polymerase which incorporates labelled nucleotides using the hybridized probe as a primer and the cellular DNA or RNA as the template (the labelled nucleotide is represented by a black dot in the diagram). The target is directly visible under the microscope if fluorochrome-labelled nucleotides have been incorporated or can be visualized with fluorochrome labelled antibodies or avidin if haptenlabelled (digoxigenin or biotin) nucleotides have been used.

Primer

dNTP

DNA polymerase

principle

Chromosome

5'

3' \

Fig. 1. The principle of PRINS

M. Clark (Ed.), In Situ Hybridization.0 Chapman & Hall, Weinheim. ISBN 3-8261-0038-7

variants of PRINS method

PRINS vs. FISH advantages of PRINS signal to noise ratio speed

specifity and sensitivity in analysis of minor sequence variations

We are developing several variants of the PRINS method: Cyclic PRINS reactions with one primer or multiple independent primers giving a linear amplification of the labelling (repeated-PRINS), cyclic PRINS reactions with primer sets which can give exponential amplification by the formation of labelled PCR-products (PRINS-PCR), and finally PRINS performed on in situ pre-amplified DNA or RNA (amplified by PCR using unlabelled nucleotides) (PCR-PRINS). The PRINS variants based on temperature cycling are all developed in order to achieve increased sensitivity by formation of multiple copies of the target sequence in situ. Due to the fact that the probes used for the various PRINS alternatives are unlabelled, high concentrations can be used without giving rise to background signals; probe bound non-specifically to structures in the cell will not cause labelling since only correctly hybridized probe can function as primer for chain elongation. The ability to use high probe concentrations makes the hybridization very fast. A PRINS reaction (denaturation, annealing and chain elongation) can be ready for evaluation in 10-30 min when using fluorochrome-labelled nucleotides, and in about 1-2 hours when using hapten-labelled nucleotides. The speed of the PRINS reaction minimizes damage to tissue structures. Thus good preservation of cellular morphology and high quality banding of chromosomes is possible. In many situations specific detection of minor sequence variations (e.g. somatic mutations) in situ is required. Traditional in situ hybridization can to some extent achieve this by using a non-radioactive labelled allele-specific oligonucleotide as probe. Two problems are, however, encounted when using this technique. Firstly, the discriminatory power of an allele-specific oligonucleotide is not as high as in priming assays, where the discrimination is based on hybridization and subsequent chain elongation of the probe (Syvanenand Landegren, 1994). Secondly, the sensitivity achieved by FISH with allele specific oligonucleotides is inherently low, due to the fact that only a few reporter molecules can be contained within each oligonucleotide. Since PRINS is based on specific hybridization and initiation of chain elongation, high discriminatory power is achieved. At the same time, sensitivity is high since a multitude of reporter molecules can be incorporated during chain elongation, making the signal independent of primer length. Furthermore, the sensitivity of PRINS can be increased by temperature cycling. In this chapter we present the protocols for :

1. Standard DNA-PRINS reactions with oligonucleotide probes DNA-PRINS with probes obtained by restriction enzyme digestion of cloned DNA ( H i n d k j a et al., 1991, Koch et al., 1991)

2. Detection of multiple probes in different colours (multicolourPRINS)(Hindkizret al., 1994)

3. A combination of PRINS and chromosome painting (PRINSpainting)(Hindkiaet al. 1995) J. HINDKJAR eta/.

4. A temperature cycling PRINS method for D N A targets (Terkelsen etal., 1993)

5. A temperature cycling PRINS method for the detection of mRNA.

3.2 Materials and Chemicals 3.2.1 Buffers and Reagents Blocking solution for DNA: 5 % non-fat dry milk dissolved in washing buffer. Centrifuge for 2 rnin in an Eppendorf centrifuge and use supernatant. Blocking solution for mRNA: 1 % Blocking reagent (Boehringer Mannheim) dis-

solved in buffer 1. Buffer 1: 100 mM Tris-HCI, pH 7.5, 150 mM NaCI. Buffer 2: 100 mM Tris-HCI, pH 8.8, 100 mM NaCI, 5 mM MgCI:, Fast red solution: 2 mg N-AS-Mx (Naphtol As-Mx phosphate) is dissolved in 0.2 ml DMF (dimethylformamide)and transfered to 9.8 ml Tris-HCI (0.1M) pH 8.2. This solution is stored at -20 "C. Dissolve 1 mg Fast red (Sigma)in 1 ml in the solution above and filter through a 0.45um millipore filter. High-salt-buffer: 1.5 mM MgCI2,550 rnM KCI and 1500 mM Tris-HCI, pH 8.3.

PBS 130mM NaCI, 7mM Na2HP04,3mM NaH2P04,pH 7.4 Proteinase K (5 pg/ml in lOOrnM Tris-HCI, pH7.5, 50 mM EDTA

lox Reverse transcriptase buffer: 500 mM Tris-HCI, pH 8.3, 750 rnM KCI, 30 mM MgCI:,.

RPMl 1640 supplemented with 10% foetal calf serum 20x SSC (3 M NaCI, 300 mM sodium citrate, pH 7.0) Stop buffer: 50 rnM NaCI, 50 mM EDTA, pH 8.0. 10xTBE: 900 mM Tris-base, 900 mM boric acid, 1 rnM EDTA.

lox Taq polymerase buffer: 500 rnM KCI, 100 mM Tris-HCI, pH 8.3, 15 mM MgCI:,, 0.1 % (W/V) BSA (or gelatine). Washing buffer: 4x SSC, pH 7.0,0.05% Tween 20. ( l x SSC: 150 mM NaCI, 15 mM

sodium citrate).

3.2.2 Chemicals Anti-digoxigenin, monoclonal antibodies Anti-digoxigenin-fluorescein, Fab fragments Anti-fluorescein, monoclonal antibodies Antifade mountant : Vectrashield Biotin-ll-dUTP Biotinylated anti-Avidin D antibodies DAPl Dde I, restriction enzyme Dideoxy-nucleotidemixture: 2.5 mM of each of ddATP, ddCTP, ddGTP and ddlTP

Boehringer Mannheim

1333062

Boehringer Mannheim

1207741

Boehringer Mannheim

1426320

Vector Laboratories Sigma Vector Laboratories

H 1000 86780 BA0300

Sigma Boehringer Mannheim Boehringer Mannheim

D9542 835293 775304

Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ Labelling (PRINS)

Digoxigenin-11-dUTP Ethidium bromide Fluorescein Avidin DCS Fluorescein-12-dUTP Fluorescein-conjugatedrabbit anti-mouse antibodies Fluorescein-conjugatedswine anti-rabbit antibodies Foetal Calf serum Formamide Glycerol, 87 % Nucleotides: dATP, dCTP, dGTP and d l l P Propidium iodide Proteinase K Rhodamine-dUTP Sheep-anti-rabbit Superscript RNase H Reverse transcriptase Taq DNA polymerase

1093088

Boehringer Mannheim Sigma Vector Laboratories Boehringer Mannheim Dako

E7637 A2011 1373242 F261

Dako

F205

Gibco BRL Merck Merck Boehringer Mannheim

10081 103264R 101184K 1277049

Sigma Boehringer Mannheim Boehringer Mannheim Sera Lab Gibco BRL

P4170 161519 1534378 Sld 2507 18053-017

Boehringer Mannheim

1146165

3.2.3 Equipment

Staining jars. Thermo-block. Water-bath with humidified chamber. Thermal cycler (Hybaid). DNA gel electrophoresis apparatus. Fluorescence microscope.

3.3 DNA-PRINS with Oligonucleotide Probes

probe design -forsPecificDNAfamilies

- for repeated DNA families

J. HINDKJAR etal.

To detect a specific DNA target in chromatin by PRINS, a suitable oligonucleotide probe can be designed. This is achieved by studying DNA sequences found in universal databases, and selecting a sequence specific for the target DNA in question. The design of primers for the detection of single copy genes is normally not a problem since these DNA sequences are usually unique. However, it must be checked that no significant homology exists to other genes or particularly to repeated sequences. Design of specific probes for repeated DNA families, such as the centromeres of human chromosomes, is more troublesome since the DNA sequences within different members of a repeat family possess a high degree of homology which can give rise to cross hybridization of the probe. This is a well known problem when performing FISH, where hybridization must be performed with high stringency to avoid cross hybridization. However, when studying corresponding sequences from different chromosomes carefully, major differences in short stretches of the sequence can be found. Synthesising an oligonucleotide representing such a small unique sequence within the repeat makes it possible to perform PRINS reaction with specific detection of a chromosome pair without cross hybridization even at low stringency.

We have designed oligonucleotide probes specific for almost all human chromosomes (Koch et al., 1995). The length of the oligonucleotides we design is usually between 16-45 bases. The theoretical melting temperature of the oligonucleotide can be used as a guideline for the reaction temperature used. However, a titration of temperature and concentration of the oligonucleotide probe should be performed to find the optimal reaction conditions. Normally, 0.1 -2 pg of DNA/SOpI of reaction mixture is used.

determine optimal reaction temperature

1. The PRINS reaction mixture is prepared by mixing :

DNA-PRINS (oligonucleotide probes)

Mix gently by tapping a finger on the Eppendorf tube. It is often easier to add H 2 0first to ensure that the small volumes of the other reagents are totally dispensed into the reaction mixture. Glycerol helps to avoid evaporation of the reaction mixture during incubation. The unlabelled nucleotides, Taq polymerase buffer, glycerol, and H 2 0can be mixed and stored at -20 "C in aliquots of 40 pI in an eppendorf tube. When a PRINS reaction is to be performed then DNA, labelled nucleotide, Taq polymerase and H 2 0are added to a final volume at 50 pl. The PRINS reaction mixture is now ready for use.

2. Preheat the slide with the metaphase chromosomes on a thermo-block at 94 "C for about 15 s before adding the reaction mixture which is then spread and covered with a 25x50mm coverslip. Denature the chromosomal DNA by heating the slide for 4 min at 94 "C.

3. After denaturation the slide is quickly transferred to a humidified chamber at the stringent temperature for primer annealing and chain elongation. Incubate for 2-60 min before stopping the PRINS reaction by washing the slide in 100 ml of preheated stop buffer in a staining jar for 1 min at annealing temperature. Taq DNA polymerase has been successfully used in PRINS employing a 16-mer oligonucleotide at the relatively low temperature of 30 "C, and may work at even lower temperatures. 4. Transfer the slide to 50 ml of washing buffer in a staining jar with screw-cap, and wash with gentle agitation for 3 min at room temperature. The slide is now ready for visualization (see section 3.9.1).

low temperature chain elongation

Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ Labelling (PRINS)

This method of DNA- PRINS is illustrated in fig 2. This shows the labelling of the X-chromosomes of a human female metaphase using a 35-mer oligonucleotide specific for the alpha satellite DNA subfamily on the X chromosome as a primer and digoxigenin-11-dUTP as the labelled nucleotide (Fig. 2).

3.4 PRINS with Ddel Digested Cloned Probes Cloned probes specific for the alpha satellite DNA of centromeres are available for all human chromosomes (Choo et al., 1991).The alpha satellite DNA is composed of higher order repeats each containing a Fig. 2. PRINS with a 35-mer oligonucleotide primer (5'-CCTGAAAGCCTTTT- b CGCTTTATCTTCACAGAAAGA-3') specific for the alpha satellite D N A of the human X chromosome. Digoxigenin-11-dUTPis used as the labelled nucleotide and visualized with fluorescein conjugated anti-digoxigenin Fab fragments. The PRINS reaction was performed for 15 min at 60 "C. Chromosomes are counterstained with propidium iodide

Fig. 3. Multicolour-PRINS with three oligonucleotide probes representing the alpha satellite DNA of chromosome 18 (Y-CGTTTCAAAACTTCTCTATGAAAAGAAAGGTTCTACTCCmA-3'), alpha satellite DNA of chromosome 10 (5'CTAGTTCTACGAGACTCTGTGTCGAGCTCGACTTTT-3') and satellite Ill DNA of chromosome 9 (5'-CCATTCCAllCCAYTCGGGTT-3'),respectively. First PRINS of chromosome 18 was performed at 60 "C for 30 min with digoxigenin-11-dUTP as the labelled nucleotide and visualized with AMCA conjugated anti-digoxigenin Fab fragments. Secondly PRINS of chromosome 10 was performed at 63 "C for 30 min using biotin-11-dUTPand visualized with fluorescein conjugated avidin. Finally PRINS of chromosome 9 was performed at 65 "C with rhodamin-dUTP which is directly visible After microscopical analysis of the three probes, the slide was washed and the chromosomes counterstained with DAPI. The same metaphase was found and a new picture was taken of the metaphase. Pictures of probes and metaphase cell were combined in a computer program IPLab from Signal Analytics (Virginia, USA) Fig. 4. PRINS-paintingof a metaphase cell from a patient with chromic myelogenous leukaemia (CML) with a reciprocal translocation between chromosomes 9 and 22 t(9;22)(q34;qll). Chromosome 9 was detected by PRINS using an oligonucleotide representing the satellite 111 DNA situated in the centromeric region of chromosome 9 (see sequence in legend for figure 3).The reaction was performed at 65 "C for 10 min with rhodamin-dUTP as the labelled nucleotide. After washing and dehydration, a biotin-labelled chromosome painting library representing chromosome 22 was applied to the slide and hybridized overnight at 37 "C. The painting library was visualized with fluorescein-conjugated avidin. Chromosomes were counterstained with DAPI. The satellite Ill DNA of the normal chromosome 9 and the der(9) are stained in red. The translocated DNA material from chromosome 22 of der 9 is stained in green with the chromosome 22 library (arrow). The normal chromosome 22 (large arrowhead) and the der(22)(Philadelphio chromosome)(small arrowhead) are also stained with the chromosome 22 library. Since DNA libraries of acrocentric chromosomes contain DNA sequences shared among all acrocentric chromosomes signals are also seen in the satellite region of these chromosomes Fig. 5a,b. PRINS-PCR using a primer set specific for the alpha satellite DNA of human chromosomes, with fluorescein-12-dUTP as the labelled nucleotide, performed on chromosomes from a human-hamster cell line (ALJ2) (a) 1 cycle (b) 20 cycles Fig. 6. Repeated-PRINS using two primers representing a motif in kringle 4 of the apo(a) gene situated on chromosome 6. 20 cycles were performed using digoxigenin-11-dUTPas the labelled nucleotide. The signal was visualized with fluorochrome conjugated anti-digoxigenin Fab fragments. The signal was seen on both chromosomes 6 (arrows) and in the interphase nucleus (arrowheads) J. HINDKJAR eta/.

Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ Labelling (PRINS)

specific number of monomers with an average length of 171 base pairs. Each monomer consists of a conserved and a variable region. A motif in the conserved region is recognized by the restriction enzyme Ddel, which can therefore cleave higher order repeats into the 171 base pair monomers. The resulting monomers can be used as primers for PRINS (Koch et al., 1991). The differences in the variable region of the monomers among alpha satellite DNA subfamilies from different chromosomes make it possible to discriminate the centromeres of these. Thus, performing a high stringency PRINS reaction with a Ddel digested cloned alpha satellite DNA probe, strong chromosome specific labelling of a chromosome pair can be obtained. The specificity and sensitivity is similar to that obtained using a nick translated cloned probe for FISH, but the PRINS technique is faster.

3.4.1 Generation of Primers from Cloned Alpha Satellite DNA 1. Digest 100 pg of cloned alpha satellite DNA with the restriction enzyme Ddel(1 U/pg DNA) in a total volume of 100 pI for 2-3 h at 37 "C. probe generation

check probe homogenity

2. Run 1 pg of the digested DNA on a 2 % agarose gel prepared in l x TBE to check that the DNA is totally cleaved. As a control, run 1 pg of the undigested DNA probe and a marker DNA with fragments in the region of 150-200 base pairs in parallel. 3. Following electrophoresis the gel is stained in ethidium bromide (1 pg/ml) for 10 min and rinsed for 5 min in distilled water. Evaluate the result under UV. If the DNA probe is digested, it is now ready for use in a PRINS reaction without any purification.

3.4.2 PRINS Reaction 1. The PRINS reaction mixture contains: W DNA PRINS (probes from cloned + digested DNA)

2. The slide with the metaphase chromosomes is placed on a thermo-block and preheated for 15 s at 94 "C before the PRINS reaction mixture is added and covered with a 25x50 mm coverslip. The denaturation is performed for 4 min at 94 "C before the slide is quickly transferred to a humidified chamber at stringent temperature (70-77 "C). Here annealing and chain J. HINDKJAR etol.

elongation are performed for 15-60 min before the reaction is terminated by washing the slide in stop buffer for 1 min at annealing temperature.

3. Transfer the slide to washing buffer, and wash with gentle agitation for 3 min at room temperature. The slide is now ready for visualization (see section 3.9.1).

3.5 Multicolour-PRINS It is often very useful to be able to detect different sequences in various colours simultaneously in metaphases or interphase nuclei. This is possible with the PRINS method by performing successive PRINS reactions with primers detecting different target sequences. The sequential chain elongations are performed with differently labelled nucleotides and can thus be visualized in various colours. Since the primer extensions are performed separately the PRlNS technique offers the opportunity to combine primers with different hybridization characteristics.This means that a short oligonucleotide can be visualized together with a longer fragment from a cloned probe (Hindkisr et a/., 1994).

combinatonof various primers

1. Perform a standard PRINS reaction as described in section 3.3 if the primer is an oligonucleotide or in section 3.4 if the primer is from a cloned probe. Since chain elongation raises the melting point of the hybridized probe the shortest probe can be used first if desired, since, due to chain elongation, it will not dissociate when temperature is raised to the stringent temperature of the longer primer.

multicolour-PRINS

2. After incubation at stringent temperature for hybridization and chain elongation the slide is washed in stop buffer for 1 min at incubation temperature, followed by a dehydration in an icecold (-20 "C) ethanol series (70%, 90% and 99%, 2 min each).

3. The slide is air dried and preheated to the stringent temperature of the second probe for 30 sec in the humidified chamber. The reaction mixture of the second PRINS reaction is applied and spread with a coverslip.

4. After incubation at stringent temperature for 2-60 min the slide is washed in stop buffer for 1 min. If a third PRINS reaction is to be performed, the slide is again dehydrated, air dried and preheated to stringent temperature of the third probe before the PRINS reaction mixture is applied and the hybridization and chain elongation performed. The PRINS reaction is terminated by incubation for 1 min in preheated stop buffer and washing for 3 min in washing buffer. Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ Labelling (PRINS)

5. The slide is ready for visualization (see section 3.9.1). The different PRINS reactions should be performed with differently labelled nucleotides, which then are visualized in different colours. A multicolour PRINS using three oligonucleotides specific for the chromosomes 9, 10 and 18 is shown in Fig. 3.

3.6 PRINS-painting PRINS can be combined with chromosome painting to pinpoint the centromere (or another locus) of a specific chromosome pair. This method can be used with success as a supplement to chromosome painting with multiple probes, particularly when studying translocations. Investigation of break-points using chromosome-specific probes from the proposed translocated regions may produce ambiguous results as signal arising from very small translocations may be hidden by that from adjacent chromosome paints. When only the centromere recognition of small of a specific chromosome is labelled in addition to chromosome translocations painting with one library, then a small translocation is easier to recognize since the neighbouring DNA is unlabelled (Hindkizr etal., 1995). 1. A PRINS reaction mixture is prepared according to the procedure described in section 3.3, applied to a slide preheated to 94 "C and covered with a coverslip (remember to use a labelled PRINS-painting

J. HINDKJAR etal.

nucleotide which is different from the one to be used for chromosome painting). After a 4 min denaturation at 94 "C the slide is transferred to a humidified chamber at the stringent temperature of the probe. 2. Hybridization and chain elongation is performed for 2-60 min. The PRINS reaction is terminated by washing in stop buffer for 1 min at annealing temperature. The slide is dehydrated through an ice-cold (-20 "C) ethanol series (70%, 90 % and 99 %, 2 min each). 3. The slide is air dried and preheated at 37-42 "C for 30 sec before 10 pI of painting probe in hybridization buffer is added and covered with a 18x18 mm coverslip which is sealed with rubber cement. If the painting probe was bought commercially, follow the manufacturers instructions on how to treat the probe prior to application to the slide. The painting probe is prepared during the PRINS reaction so it is ready for application to the slide immediately after the drying and preheating of the slide. 4. The hybridization is performed overnight at 37 "C-42 "C. The next morning the rubber cement is removed and the slide is washed as follows : twice for 10 min in 50% formamide, 2xSSC at 42 "C twice for 5 min in 2xSSC at 42 "C. The slide is now ready for visualization (see section 3.9.1).

A PRINS-painting of a translocation (9;22)is shown in Fig. 4. The PRINS reaction is performed with a chromosome 9-specific oligonucleotide and the chromosome painting with a chromosome 22 specific DNA library.

3.7 PRINS-PCR and Repeated-PRINS of DNA A flow diagram for the steps in PRINS-PCR is shown in Fig. 7 a, b below. For PRINS-PCR of DNA two model systems were used for the development of this technique.

+

In situ cDNA synthesis

Pretreatment

Temperature-cycling

I-=[

[TI Fixation

Immunostaining

+

Wash

L

Mounting

Fig. 7. Flow diagram illustrating the steps in PRINS-PCR of chromosome DNA (left column) and PRINS-PCR of cytoplasmic mRNA (right column) using micro-slides

Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ Labelling (PRINS)

Firstly, cyclic labelling methods were used on the alpha-satellite

DNA repeat family situated in the centromeric region of all human chromosomes (Terkelsen et al., 1993).This system was used for the development of PRINS-PCR. The two primers used were derived from the conserved region of the alpha satellite DNA monomer and located in such a way that during amplification, a product of at least 171 base pairs is generated. The primer sequences are: Primer A (5’-CATCACAAAGAAGTTTCTGAGAATGCTTC-3’) Primer B (5’-TGCATTCAACTCACAGAGTTGAACCTTCC-3’) In this series of experiments, a hybrid human-hamster cell line ALJ2 (Waldren et al., 1984),containing chromosome 11 as the only human chromosome, was used as the target cell. The use of this hybrid cell line allowed us to utilize the hamster chromosomes as negative controls to determine the signal-to-noise ratio. The second system was based on detection of the gene apo(a), which contains 10-40 copies of a 342 bp kringle IV motif (McLean et al., 1987).Here, also, two primers were used, but in this case the distance between them was more than 8 Kb. This meant that only repeated-PRINS (linear amplification) was expected to occur in this system. Primer C (5’-CCCAGGCCTTTGCTCAGTCGGTGC-3’) Primer D (5’-GGGTGCAGGAGTGCTACCATGGTAATGGAC-3’) Human lymphocytes were used as test cells. In the human genome apo(a) is located very distally on chromosome 6 (6q26-27).

3.7.1 Chromosome Spreads

cyclic PRlNS reactions

Micro-slides, 2 mm wide and 20 mm long, were cut from standard cover slips. Metaphase chromosomes fixed in methanol and acetic acid (3:l) were spread on the micro-slides by conventional methods. The micro-slides were then air dried, and placed inside an Eppendorf tube (Figure 8).

Fig. 8. Micro-slides, 2mm wide and 20 mm long, cut from standard coverslips J. HINDKJAR etal.

3.7.2 Pretreatment of Chromosome Spreads 200p.1of a high-salt-buffer (1.5 m M MgCI2, 550 m M KCI and 1500 mM Tris-HCI, pH 8.3) is added to the eppendorf tubes. The tubes are then heated to 94 "C for 10 to 30 minutes in a Hybaid Thermal Cycler. The slides are dehydrated through an ice-cold ethanol series (70%, 90% and 99% ethanol, 5 minutes each). The microslides are air dried and placed in new eppendorf tubes.

3.7.3 labelling Reaction 1. The reaction mixture is prepared by mixing:

The reaction mixture is added to the eppendorf tube containing the microslide treated with high salt buffer. The MgCI2 concentration is very important for the PCR reaction. Therefore the MgCI2should be titrated to find the optimal concentration.

2. PRINS-PCR with alpha-primers (primer A and B): First cycle: denaturation step at 94 "C for 4 minutes annealing and chain elongation for 10 min at 70 "C. Performed once and linked to second cycle. Second cycle: denaturation for 1 min at 94 "C annealing and chain elongation for 10 min at 70 "C. In total 16 or 20 cycles were usually performed. Performing more than 20 cycles usually destroyed the chromosomes. Linked to third cycle. Third cycle: No denaturation, only elongation at 70 "C for 7 minutes.

3. Repeated-PRINS of kringle IV: First cycle: denaturation for 4 min at 94 "C annealing for 3 min at 63 "C chain elongation for 6 min at 70 "C. Linked to second cycle.

determine optimal MgClz concentration

@

PRINS-PCR

@

repeated PRINS

Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ Labelling (PRINS)

Second cycle: denaturation for 1 min at 94 "C annealing for 3 min at 63 "C chain elongation for 6 min at 70 "C. Normally 10-20 cycles are performed. Linked to third cycle. Third cycle: N o denaturation, only elongation at 70 "C for 7 minutes.

4. The micro-slides in both cases are washed in eppendorf tubes with washing-buffer for 2x15 min and the reporter molecules are visualized (see section 3.9.2).

5. After visualization the micro-slides are placed face-up on a standard slide and mounted in antifade solution containing propidium iodide (0.5 p.g/pI), and covered with a 25x50 cover slip. The slides are analyzed by conventional fluorescence microsCOPY,

Using PRINS-PCR with alpha satellite primers, a distinct signal is visible after a few cycles. However, when more cycles are performed the signal spreads outside the chromosome, first in a butterfly shape and finally as vast amounts of PCR product covering the whole chromosome (Fig. 5a,b). When the amount of accumulated product was quantified, using confocal laser scanning microscopy, it becomes evident, that mostly linear amplification occurs during the first 10-15 cycles, and that true PCR amplification only occurs when the product appears outside the boundaries of the chromosome (Terkelsen eta/., 1993). On the contrary when repeated-PRINS is performed - as with the apo(a)-primers - signal stays localized for at least 20 cycles resulting in very distinct spots (Fig. 6). In most situations we would therefore recommend the repeated-PRINSversion since the sensitivity seems sufficient even for unique sequences and the localization of signal is much better preserved.

3.8 PRINS-PCR of m R N A PRINS of mRNA was developed in our laboratory (Mogensen eta/., 1991). We have, however, had difficulties with this method, mainly due to severe non-specific priming. We have therefore recently turned to a reverse transcriptase-based PRINS-PCR of mRNA assay (Fig. 7). This assay is based on initial synthesis in situ of first strand cDNA followed by.cyclic PRINS of this with sequence-specific primers. This method has turned out to be a very sensitive assay for in situ detection of specific mRNA and may well be developed to a point, where even single nucleotide exchanges can be detected. The model system used for the development of PRINS-PCR of RNA is the labelling of the mRNA coding for the fatty acid degradation enzyme medium chain J. HlNDKJkR etal.

acyl CoA dehydrogenase (MCAD) in cultured human fibroblasts. MCAD is a housekeeping enzyme present in low amounts in all cell types. Thus normal FISH detection barely possible. The primers used are a set developed for the clinical diagnosis of an abundant diseasecausing point mutation (A985G) in exon 11 of the gene: sense primer: 5'-TTTATGCTGGCTGAAATGGCCATG-3', a nt isense primer : 5' -AGTTTTTCTACAG GATATTCTGTATTAAATCC-

ATGGCCTC-3'. This primer set amplifies a fragment of 220 b p and works efficiently on naked DNA. Both the in situ cDNA synthesis and the following cycling reaction are performed on the same micro-slides as used for repeated-PRINS on chromosomes. Fibroblasts for the assay are initially grown to confluence on such micro-slides in Petri dishes.

1. For the detection of cytoplasmic mRNA, skin fibroblasts from a healthy individual were cultured on the micro-slides in standard RPMl 1640 medium supplemented with 10 % foetal calf serum in Petri dishes.

2. After a short wash in PBS, the cells were fixed in 4 % paraformaldehyde/O.l % glutaraldehyde for 5 min on ice and washed twice for 5 min in PBS. The slides were stored in 70 70ethanol at -20 "C until required.

3. Before starting PRINS-PCR, the micro-slides were briefly washed in PBS and subjected to the following pretreatment:

4. Slides are then blocked for 30 minutes at room temperature with blocking solution (for mRNA), and finally briefly washed in PBS.

5. The micro-slides are then transferred to a standard PCR tube preheated to 50 "C and cDNA synthesis is performed in 100 pI reaction mixture for 60 minutes at 50 "C. The cDNA reaction mixture contains:

Final reaction volume of 100 pI of reverse transcriptase buffer. Detection of Nucleic Acids (DNA and RNA] In Situ by Single and Cyclic Primed In Situ Labelling (PRINS)

6. The micro-slides are then transferred to a new tube containing PBS at room temperature. The PRINS-PCR reaction is performed in a new tube containing 100 pl of PCR reaction mixture. Reaction mixture:

Final reaction volume of lOOyl of Taq polymerase buffer.

7. The tube is placed in an Omnibed PCR cycler and 10-20 cycles are performed with the following program: First cycle: Denaturation at 94 "C for 8 minutes Annealing for 2 minutes at 61 "C Elongation at 74 "C for 2 minutes. Linked to second cycle. Second cycle: Denaturation at 94 "C for 2 min Annealing for 2 minutes at 61 "C Elongation at 74 "C for 2 minutes. Linked to third cycle.

Third cycle: No denaturation, only elongation for 7 min.

8. After the last cycle the micro-slide is washed very briefly in PBS and then fixed in 4 % paraformaldehyde for 5 minutes on ice. The slide is then washed 2 x 2 minutes in PBS. The slide is now ready for visualization (see section 3.9.2). In a separate series of experiments, we instead added 100 pl paraformaldehyde directly to the PCR-tube before removing them from the Omnibed PCR Cycler. After 30 minutes the micro-slides were then transferred to PBS and washed 2 x 2 minutes. This procedure did not alternative reaction give rise to notably higher background, but there was no apparent stopping and fixation increase in signal either, so this step was omitted. The result of a cycling reaction using this protocol is shown in Fig. 9. It is evident, that 20 cycles with two primers results in strong cytoplasmatic staining. Attempts to quantify this cytoplasmatic staining have evaluation of results been made after fluorescence labelling. Substantial cell to cell differences make these measurements questionable, but it was our impression that the cycle-dependent increase in signal was at best linear. However, the fact that 20 cycles with only one primer results in less than half as much signal than 20 cycles with two primers does suggest that some PCR amplification occurs. We are therefore of the opinion, that the signal reflects a rather low efficiency PCR amplifica-

+R

J. HlNDKJPiR eta/.

Fig. 9 a-d. Detection of MCAD mRNA using PRINS-PCR and the protocol described. (a) 20 cycles, (b) 10 cycles, (c) 1 cycle, (d) 20 cycles (of only the sense primer]. Cells were stained with the Fast Red method for micro-slides described in section 3.2

tion, and thus recommend the use of a set of PCR primers for maximally sensitive detection of mRNA by this method. If subcellular localization is mandatory then repeated-PRINS using one primer can be considered.

3.9 Visualization of Hapten-labelled Nucleotides 3.9.1 Standard Slides 3.9.7.7 Biotin Perform all incubations and washes at room temperature

1. Drain the slide and apply 100 pI of blocking solution. Incubate for 5 min under a coverslip.

2. Wash the slide for 15 sec in 50 ml of washing buffer. Drain and add 100 pI of fluorescein avidin in blocking solution (2 ng/pl), and incubate for 10-30 min. 3. Wash 3x5 min in 50 ml washing buffer using gentle agitation. 4. Mount the slide in 20 pI antifade solution containing propidium iodide (0.5 pg/ml) or DAPl (0.15 pg/ml). Evaluate the result

@

protocols

under a fluorescence microscope. Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ Labelling (PRINS)

enhanced staining

If staining is too weak, apply additional layers of fluorescein. First wash the slide for 2x 5 min in washing buffer. Add 100 y1 of anti-avidin antibodies in blocking solution (2 ng/yI) and incubate for 10-30 min. Wash 3x 5min in washing buffer. Add 100 y1 of fluorescein avidin in blocking solution (2 ng/yI) and incubate for 10 min. Wash for 3x5 min in washing buffer. Mount in antifade solution containing propidium iodide or DAPl and evaluate under the microscope. Similar procedures are used for visualization with rhodamine- or AMCA-coniugated avidin.

3.9.7.2 Digoxigenin Perform all incubations and washes at room temperature

1. Drain the slide and apply 100 yl of blocking solution and incubate for 5 min.

2. Wash the slide for 15 sec in 50 ml of washing buffer. Drain and add 100 pl of anti-digoxigenin-fluorescein, Fab fragments in blocking solution (2 ng/pI) to the slide and incubate for 10-30 min under a coverslip.

3. Wash 3 x 5 min in 50 ml of washing buffer using gentle agitation. Drain the slide and mount in 20 yI of antifade solution containing propidium iodide (0.5 yg/ml) or DAPl (0.15 yg/ml) and evaluate the result under a fluorescence microscope.

enhanced staining

If staining is too weak, apply additional layers of fluorescein. First wash the slide for 2 x 5 min in washing buffer and drain. Add 100 yI anti-fluorescein antibodies (mouse) in blocking solution (5 nglyl), and incubate for 30 min followed by a 3 x 5 min wash in washing buffer. Add 100 yI of fluorescein-coniugated rabbit anti-mouse antibodies (diluted 1 :lo0 in blocking solution), and incubate for 30 min followed by a 3 x 5 min wash. Mount in antifade solution containing propidium iodide or DAPI, and evaluate under the microscope. If signal is still too weak, apply an additional layer of fluorescein conjugated antibodies. Wash the slide for 2 x 5 min in washing buffer. Add 100 pI fluorescein-conjugated swine anti-rabbit antibodies (diluted 1 :lo0 in blocking solution), and incubate for 30 min followed by a 3 x 5 min wash. Mount in antifade solution containing propidium iodide or DAPl and evaluate under the microscope. Rhodamine- or AMCA-coniugated anti-digoxigenin antibodies can also be used for visualization. However, the amplification with additional layers described above is for fluorescein conjugated antidigoxigenin onIy.

J. HINDKJAR etol.

3.9.2 Micro-slides 3.9.2.7 Detection of Labelled DNA The procedure for detection of digoxigenin or biotin is the same as described above for standard slides, except that incubations and washes are performed in Eppendorf tubes.

3.9.2.2 Detection of labelled RNA We use either alkaline phosphatase-conjugated anti-digoxigenin Fab fragments, followed by development with the fast red system or fluorescein-conjugated anti-digoxigenin antibody for detection of labelled RNA.

1. The microslide is blocked for 60 min in 1 % blocking solution (120pVtube).The slide is transferred to an anti-digoxigenin antibody (monoclonal mouse antibody) diluted in 1 % blocking solution (0.5 ng/pl) and incubated for 60 min at room temperature. Visualization can then be performed using either an alkaline phosphatase-conjugated or fluorescein-conjugated antibody.

3.9.2.3 Alkaline Phosphatase Visualization 1. Wash the micro-slide 3x5 min in buffer 1. 2. Incubate the slide with alkaline phosphatase conjugated rabbit anti-mouse antibodies antibody (diluted 150 in 1 % blocking solution) (100 $/tube) for 60 min at room temperature.

3. Wash 3x5 min in buffer 1. If a stronger signal is required, the slide can now be incubated with sheep anti-rabbit antibodies (diluted 150) in 1 % blocking solution) (100/tube) for 60 min at room temperature. Wash 3x5 min in buffer 1. 4. The slide is now rinsed in buffer 3 before it is transferred to the fast red solution where it is incubated for up to 20 min in the dark at 37 "C to develop the signal. The reaction is stopped by washing with distilled water.

5. The micro-slides are placed on a standard slide (can be glued to the slide with Eukitt) and covered with a 25x50 mm coverslip. The preparation is now ready for microscopy.

Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ Labelling (PRINS)

3.9.2.4 Fluorescent Visualization 1. Wash the micro slide 3x5 min in PBS. 2. Incubate the slide in flourescein conjugated rabbit anti-mouse antibodies (diluted 1 :lo0 in 1 % blocking solution) (100yl/tube) for 60 min at room temperature.

3. Wash 3x5 min in PBS. If stronger signal is required, the slide can be incubated in flourescein-conjugated swine-anti rabbit antibodies (diluted 1 : 100) in 1 % blocking solution) for 60 min at room temperature. Wash the slide 3 x 5 min in PBS.

4. The micro-slide is placed on a standard slide, an antifade solution is applied and the slide covered with a 25 x50 mm coverslip. It is now ready for microscopy.

3.10 Troubleshooting No signal obtained using a newly synthesized oligonucleotide:

troubleshooting

hybridization temperature was too high : Make a titration of temperature to find the optimal hybridization conditions. concentration of primer was too low : The amount of primer used in the reaction mixture should also be titrated. Normally, 0.1 -2yg of primerh0yI of reaction mixture is used. The signal is hardly recognisable due to too much background staining: The hybridization temperature was too low : raise the temperature. Too much primer was used : Make a titration of primer concentration. *The chromosome preparation was of poor quality, Old chromosome spreads often contain nicks, which can function as primers for chain elongation. Chromosome spreads should preferably be prepared immediately before use. Slides only a few weeks old can normally be used without problems, but the older they get; the worse, the background problems. Old cell suspensions in fixative (methanol: acetic acid) can be spread and normally used without background problems. Thus it is the age of the chromosome spread and not the age of the cell preparation that gives rise to background signals. Absence of signal using double-stranded probes: Incomplete enzyme digestion of probe DNA: Check on a gel that the Ddel digestion has been successful. Interference of PRINS reaction by restriction enzyme buffer: The restriction enzyme digested probe DNA is used without removal of either buffer or enzyme. Therefore to minimize interference in the PRINS reaction, the volume of the probe DNA should not exceed 10 % of the reaction mixture.

J. HlNDKJARetal.

The signal is either too strong or too weak: Choice of detection system: Substitute a different hapten-labelled nucleotide. We have tested several different hapten-labelled nucleotides that work in PRINS eg. DIG-, biotin- and fluorochrome-labelled (fluorescein, rhodamine) dUTP. The strongest signal is obtained using OIG-labelled dUTP closely followed by that of. biotin-dUTP. Fluorochrome-dUTP produces a somewhat weaker signal. However, if a probe results in a very strong signal, it can be advantageous to use fluorochrome-labelled dUTP, since results are obtained faster and in fewer steps. 0

Double labelling of a primer used in the multicolour reaction: 0 This is observed when the primer used in the first PRINS reaction is further labelled (with another hapten) during the subsequent PRINS reaction: This problem can be solved by performing a dideoxy reaction between the PRINS reactions (Hindkiaer et al, 1994). When the slide has been dehydrated, a reaction mixture containing dideoxynucleotides, Taq polymerase buffer and Taq polymerase is applied to the slide and incubated for 15 minutes at 60 "C. The slide is washed in stop buffer and dehydrated through an ethanol series. It is now ready for the next PRINS reaction.

Weak or missing signals with PRINS-PCR and repeated PRINS: Sub-optimal Mg2+concentration and primer annealing temperature: Optimal PCR conditions can intially be established on purified DNA in standard PCR reactions. These conditions are then used as a starting point for establishing optimal parameters in the corresponding PRINS- PCR reactions. It is however, our general experience, that the optimal annealing temperature and Mg2+ concentration are not always identical in the two systems.

0

Acknowledgements This work was suppoted by grants from the Danish Cancer Society and the Danish Centre for Human Genome Research. We thank Dr. Serren Pedersen, Institute for Human Genetics, Aarhus University, for providing the chromosome 22 painting library.

References Choo, K. H., Vissel, B., Nagy, A., and Kalitsis, P. (1991)A survey of the genomic distribution of alpha satellite DNA on all the human chromosomes, and derivation of a new consensus sequence. Nucl. Acids Res. 19, 1179-1182. Hindkja, J., Koch, J., Mogensen, J., Pedersen, S., Fischer, H., Nygaard, M., Junker, S., Gregersen, N., Kerlvraa, S., Therkelsen, A. J., and Bolund, L. (1991) Primed In Situ labelling of nucleic acids. Biotech Forum Europe 12, 752-756. Hindkiter, J., Koch, J., Terkelsen, C., Brandt, C.A., Kralvraa, S., and Bolund, 1. (1994) Fast, sensitive multicolor detection of nucleic acids by PRimed IN Situ labeling (PRINS).Cytogenet Cell Genet 66, 152-154. Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ Labelling (PRINS)

Hindkiier, J., Brandt, C.A., Koch, J., Lund, T.B., Kerlvraa, S., and Bolund, L. (1995) Simultaneous detection of centromere-specific probes and chromosome painting libraries by a combination of primed in situ labelling and chromosome painting (PRINS-painting).Chromosome res 3,41-44. Koch, J. E., Kalvraa, S., Petersen, K. B., Gregersen, N., and Bolund, L. (1989) Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ. Chromosoma 98, 259-265. Koch, J., Hindkiier, J., Mogensen, J., Kalvraa, S., and Bolund, L. (1991)An improved method for chromosome-specific labeling of alpha satellite DNA in situ by using denatured double-stranded DNA probes as primers in a primed in situ labeling (PRINS) procedure. GATA 8, 171-178. Koch, J., Mogensen, J., Pedersen, S., Fischer, H., Hindkiier, J., Kalvraa, S., and Bolund, L. (1992) Fast one-step procedure for the detection of nucleic acids in situ by primer-induced sequence-specific labeling with fluorescein-12-dUTP Cytogenet. Cell. Genet. 60, 1-3. Koch, J, Hindkiier, J., Kalvraa, S., and Bolund, L. (1995) Construction of a panel of chromosome specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ. Cell Genet Cytogenet. 71, 142-147 McLean, J.V., Tomlinson, J.E., Kuang, W-J., Eaton, D.L., Chen, E.Y., Fless, G.M., Scanu, A.M., and Lawn, R.M. (1987) CDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature 330, 132-137. Mogensen, J., Kalvraa, S., Hindkjier, J., Pedresen, S., Koch, J., Nygird, M., Jensen, T., Gregersen, N., Junker, S., and Bolund, L. (1991) Non-radioactive detection of mRNA subspecies in situ by PRimed IN Situ labelling (PRINS) Expm. Res. 196, 92-98. Syvanen, A.C. and Landegren, U. (1994) Detection of point mutations by solid phase methods. Hum Mutat 3: 172-179. Terkelsen, C., Koch, J., Kalvraa, S., Hindkiier, J., Pedersen, S., and Bolund, L. (1993) repeated primed in situ labeling: formation and labeling of specific DNA sequences in chromosomes and nuclei. Cytogenet Cell Genet 63,235-237. Waldren, C., Martin, J., Sutherland, J., and Cram, S. (1984) Use of somatic cell hybrids for quantitation of mutagenesis: reduction in background mutants by fluorescence-activatedcell sorting (FACS).Cytometry 5, 584-588. Willard, H. IF, and Waye, J. S. (1987) Hierachical order in chromosome-specific human alpha satellite DNA. TIG 3, 192-198.

J. HINDKJAR eta/.

In Situ Hybridization Melody Clark Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 4

Fluorescence lmmunophenotyping and lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION): Combined In Situ Hybridization and Fluorescence lmmunophenotyping K . WE BER- MATTH IESEN

4.1 Introduction During the past twenty years rapid progress has been made in establishing powerful new tools for detecting genetic defects in tumors. Until 1970, the genetic analysis of tumors was restricted to the detection of numerical chromosome aberrations within solid-stained metaphases. After the introduction of banding techniques structural aberrations could also be detected. Later on, molecular techniques were developed, which allowed deeper insights into the molecular changes caused by the chromosome aberrations found with cytogenetic techniques. In particular Southern blot analysis, the polymerase chain reaction (PCR) and DNA sequencing have contributed to the characterization of chromosomal breakpoints and to the genes located therein. At present, fluorescence in situ hybridization (FISH) is one of the most highly regarded new techniques [Pinkel et al. 19861. A very promising variant of FISH is the so called ,,Interphase cytogenetic analysis". By using suitable DNA probes it is possible to diagnose the presence of certain chromosome aberrations from the constellation of hybridization signals in the nuclei. The most commonly used DNA probes bind specifically to repetitive centromeric sequences of certain chromosomes, allowing numerical aberrations to be detected in interphase cells. Each of the different techniques has its individual strengths. In the field of tumor genetics, for example, PCR is sensitive enough to be able to detect genetically defective cells, even if the number of tumor cells is very low. The classical chromosome analysis is less sensitive, but it is the only technique that detects all chromosome aberrations simultaneously. By means of interphase cytogenetic analysis certain chromosome aberrations can be studied in all stages of the cell cycle. Although very powerful in certain fields of research, all techniques have one disadvantage in common: With none of the techniques is it possible to unequivocally correlate particular genetic defects with particular cell populations in a given tissue. In cytogenetic techniques, for example, the cytoplasm and cell membranes of mitotic cells must be completely eliminated to obtain analyzable metaphases. Thus, all morphological and immunophenotypical details of the analyzed cells M. Clark (Ed.),In Situ Hybridization. 0 Chapman & Hall, Weinheim. ISBN 3-8261-0038-7

FICTION

principle of FICTION

K. WEBER-MATTHIESEN

are destroyed. The same problem arises in molecular genetic studies: Southern blot and PCR techniques are suitable for detecting DNA defects; however, neither technique can determine which cells in the tissue actually carry the defect. The first attempt to circumvent this dilemma was made by Teerenhovi and co-workers [Teerenhovi et al. 19841. They introduced a new technique called M A C (morphology, antibody, chromosomes) combining classical chromosome banding and immunophenotyping of metaphase cells. The advantage of the MAC-technique was that - in contrast to the classical cytogenetic analysis - chromosome banding was possible on morphologically intact metaphase cells. Cells were cultured in order to obtain metaphase stage cells, exposed to a mild hypotonic solution and then cytocentrifuged onto glass slides. The hypotonic treatment caused considerable swelling of the mitotic cells resulting in moderate spreading of the chromosomes. Since, in contrast to classical cytogenetics, the morphological structures of mitotic cells were retained by this chromosome preparation technique, immunocytochemical techniques could be applied simultaneously. It was possible for the first time to determine the immunophenotype of cytogenetically aberrant tumor cells. Unfortunately, the use of the MAC technique was quite limited for various technical reasons. Firstly, as in classical cytogenetics, only the few available metaphase cells could be analyzed. Secondly, the chromosome spreading within intact mitotic cells was usually insufficient for chromosome analysis due to considerable overlapping of the chromosomes. Thirdly, evaluating the immunophenotype and karyotype of metaphase cells was extremely time consuming. Metaphase cells had to be immunophenotyped and documented photographically; then the color of the immunophenotype had to be removed with xylene, followed by the chromosome banding procedure. Finally, the photographed cells had to be relocated under the microscope for chromosome analysis. Although this approach seemed very promising, only a small amount of data was obtained with the MAC technique [e.g. Teerenhovi et al. 1984, Knuutila and Teerenhovi 1989, Haas et al. 1987, Schlegelberger et al. 19Y0, Schlegelberger et al. 1991, Schlegelberger et al. 19941. The difficulties associated with the MAC technique have now been overcome by a new method. It is a combination of fluorescence immunophenotyping and fluorescence in situ hybridization. This technique, called “Fluorescence immunophenotyping and lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)” is the topic of this chapter [Weber-Matthiesen et al. 1992, WeberMatthiesen et al. 1993a-c, Weber-Matthiesen et al. 1995a-c]. With the FICTION technique cells can be immunophenotyped and, at the same time, be evaluated for numerical chromosome aberrations. FICTION can study all cells on a slide no matter whether they are in metaphase or in interphase. FICTION studies are possible on cytospin slides, smears, imprints and cryostat sections. A comprehensive description of the FICTION technique is given later in this chapter. At this point, however, we will give a brief introduction to the procedure to explain the principle behind the method.

Slides are fixed in acetone and then immunophenotyped by an indirect fluorescence detection system. Secondary antibodies coniugated with fluorescent dyes are used to visualize antigen-specific monoclonal antibodies. lmmunophenotyping is followed by a fixation step to preserve the fluorescent immunostaining during the harsh hybridization procedure. After fixation in situ hybridization is performed using appropriate DNA probes, for example, centromeric probes. Visualization of the hybridized probes is carried out using fluorescence-labelled antibodies. As in the well-known FISH technique, the number of hybridization signals agrees with the number of copies of a given chromosome in the cells studied. Under the fluorescence microscope the immunophenotype and the hybridization signals can be evaluated simultaneously (Fig. 1 and 2).

4.2 Application of the FICTION Method This is most effectively outlined by example: five problems (4.2.14.2.5) will be discussed facing scientists and clinicians working in tumor research and diagnostics which can be resolved using

applications

FICTION.

4.2.1 Defining the Cell Type of Chromosomally Aberrant Clones

A typical feature of Hodgkin’s disease is the presence of Hodgkin and Reed Sternberg (HRS) cells in lymph nodes of affected patients. The proportion of HRS cells in affected lymph nodes is usually very low, often far below 1 %. Because of their rarity and also because HRS-like cells also exist in other diseases, it has been doubted for a long time whether HRS cells actually represent the malignant cells in Hodgkin’s disease. In approximately 30 % of cases studied with cytogenetic techniques, aberrant clones, mostly with hyperploid chromosome numbers, were detected. With classical cytogenetic techniques it was impossible to define the cell type of the aberrant cells. They could correspond to HRS cells or to other, as yet unidentified, cell populations. This question can now be studied by means of the FICTION technique. All HRS cells strongly express the CD30 antigen, whereas the majority of the other cells in the lymphoid tissue are CD30-negative. With FICTION, cytospin slides can be immunophenotyped for CD30 and hybridized with appropriate DNA probes to show the hyperploidy typically found in cytogenetic analyses. Fig. 1 demonstrates the application of FICTION to a cytospin slide prepared from the lymph node cells of a patient with Hodgkin’s disease. lmmunophenotyping was performed for CD30 and in situ hybridization was carried out using different centromere specific DNA probes. Fig. 1 clearly shows a strongly CD30 positive Hodgkin cell containing aneuploid Fluorescence lmmunophenotyping and lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)

chromosome numbers. From a FICTION study of 30 cases of Hodgkink disease we now know that only the CD30-positive HRS cells reveal numerical chromosome aberrations, whereas CD30-negative lymph node cells regularly have normal numbers of hybridization signals [Weber-Matthiesen et al. 1995~1.

4.2.2 Defining the Differentiation State of Chromosomally Aberrant Cells Different cells of one tumor clone do not always have the same immunophenotype. In particular leukemic cells, which originate from one genetically altered immature precursor cell, mimic the various steps in the differentiation pathway of blood cells. In consequence, immature stem cells and more or less differentiated cells may coexist in the same tumor clone. All tumor cells contain the same genetic defect. If the defect is known and detectable by in situ hybridization, the FICTION technique can be used to define which differentiation states are present in the tumor clone. The immunophenotypically most immature aberrant tumor cell population probably represents the source from which the more differentiated tumor cells are derived. FICTION studies by Baurmann et al. (1993) on a case of acute myeloid leukemia (AML)with trisomy 7 showed that the trisomy was restricted to early committed (CD34-positive) progenitor cells capable of erythroid and myelomonocytic differentiation.

4.2.3 Interpretation of Ambiguous Cytogenetic Findings Clones with trisomy X are frequently found in cytogenetic analyses of hematological neoplasms. It is difficult to interpret this finding if trisomy X is the only detectable chromosome aberration. O n the one hand, the trisomy could represent the tumor clone, on the other hand it could be a so-called constitutional mosaicism. The latter is found in some phenotypically normal women carrying an extra X chromosome in a portion of cells from different tissues. In constitutional mosaicism, cytogenetic analyses of bone marrow, blood and lymph node reveal trisomic cells in all lineages of the hematopoietic system (myeloid, Bcell, T-cell, etc.). If the trisomy characterizes the tumor clone, only cells with a tumor cell associated immunophenotype are expected to be involved. By means of FICTION it is possible to distinguish whether a trisomy is due to a constitutional mosaicism or restricted to the cells of the tumor clone. This has been demonstrated in a case of malignant B-cell lymphoma recently analyzed by ourselves, in which the chromosome analysis had found only a clone with trisomy X. Fig. 2 and 3 show cytospin cells from the affected lymph node stained using the FICTION method. We showed that only cells with the tumor cell associated phenotype (kappa light chain-positive) contained three hybridization signals for the X-chromosome, whereas kappa-negative K. WEBER-MATTHIESEN

Fig. 1. A CD30-positive Hodgkin cell (red fluorescence) with three green signals for chromosome 8 and one blue signal for the Y chromosome Fig. 2. Two Kappa light-chain positive tumor cells (red fluorescence) from a B-cell lymphoma with trisomy X (three blue signals). Green signals represent the chromosome 12 (in the right tumor cell one green signal is not in focus). The hybridization signals outside the positive cells (arrows) belong to a kappa-negative cell Fig. 3. Same case as shown in Fig. 2. CD3 positiveT-cells (red fluorescence) generally showed two blue hybridization signals for the X-chromosome. In contrast, some CD3-negative cells showed the trisomy (arrows)

cells had only two hybridization signals (Fig. 2). Staining for CD3 revealed that all T-cells contained normal chromosome X numbers (Fig. 3). Thus, a constitutional mosaicism was excluded.

4.2.4 An Aid to Diagnosis The myelodysplastic syndrome (MDS) is a neoplastic bone marrow disease, which can progress towards acute myeloid leukemia (AML). Fluorescence Imrnunophenotypingand lnterphase Cytogenetics as a Tool for Investigationof Neoplasms (FICTION]

The diagnosis of MDS is sometimes difficult, particularly if the typical dysplastic alterations in the granulopoietic precursor cells are slight. In such cases, classical chromosome analysis may aid in the diagnosis by demonstrating chromosome aberrations that are associated with neoplastic disorders of the myeloid lineage. One typical chromosome aberration of myeloid neoplasm is trisomy 8. Recently, we had a case of MDS to investigate in which the clinicians were not able to make a clear diagnosis, although the cytogenetic analysis had revealed a trisomy 8. The reason was that in this case almost no signs of dysplasia were detectable by bone marrow cytology. Therefore, further indications of MDS were required before initiating a treatment that could have severe therapeutic consequences. In MDS, the malignant cells may be present in all different myeloid blood cell lineages, including granulocytes, monocytes and the precursors of erythrocytes and thrombocytes. Lymphocytic lineages, in contrast, are rarely involved. This lineage restricted involvement of tumor cells in MDS can be demonstrated by FICTION. In the above mentioned case, we were able to prove by FICTION that the trisomy 8 was present in all myeloid lineages and, moreover, in CD34-positive stem cells. Lymphocytic lineages were not affected. CD3-positive Tcells and CD20-positive B-cells always contained two hybridization signals. With the aid of the FICTION data the diagnosis of MDS could be made.

4.2.5 Overcoming the Detection limit of FISH The detection of tumor clones with aneuploid chromosome numbers by means of FISH is restricted to cases with relatively high percentages of aberrant cells. At least 1-2% of the analyzed cells must be trisomic to confirm the presence of an aberrant clone. In the case of a monosomy, more than 10% of the cells on the slide must show the chromosome loss. The reason is that normal specimens hybridized with centromeric DNA probes also show a considerable proportion of cells with one or three signals (due to nonspecific or ineffective in situ hybridization). Consequently, in samples with low numbers of tumor cells it is impossible to differentiate true aneuploidy from artificial hybridization results. This problem arises, for example, in most cases of Hodgkin’s disease (see section 4.2.1), where the malignant HRS cells rarely comprise more than 1-2 %. The same holds true if residual tumor cells after therapy have to be detected. FICTION is able to overcome this problem. True aneuploidy is confirmed, if numerical aberrations are detected exclusively within cells with tumor cellassociated immunophenotype. Hence, a low percentage of tumor cells does not limit interphase cytogenetic studies by means of FICTION, if tumor cells and normal cells have different immunophenotypes.

K. WEBER-MAllHIESEN

4.3 Materials and Chemicals 4.3.1 Buffers and Reagents PN buffer: 0.1 M NaH2P04,0.1 M Na2HP04,Ph 8

PNM buffer: Add 5 % dry milk powder to PN-buffer; heat to 50 "C overnight while stirring; add 0.03% NaN3 (poisonous); PNM buffer is stable at 4 "C for at least 6 months. Paraformaldehyde solution (1 %): Dissolve 1g paraformaldehyde powder in 60ml distilled water; add 5 drops 10N NaOH. Heat to 70-100 "C until the solution is clear. Cool down and add lOml lOOmM MgCI2.Adjust pH to 7-7.5 and make up to final volume of lOOml with distilled water. Antifade solution: To 90ml glycerol add lOml PN-buffer containing 23 mg/ml DABCO. 2OxSSC: 3M NaCI, 0.3M sodium citrate; pH 7 Hybridization mixtures: In order to ensure maximum reproducibility, all provided FICTION protocols were adapted for use with commercial DNA probes available from Oncor. These probes are supplied as biotin or digoxigenin conjugates (centromeric probes), or as mixtures of both (translocationprobes). If non-commercial DNA probes are used they should be adjusted to 10 ng/pI in TE-buffer (1 mM EDTA, 10 mM Tris-Hcl, pH8) before being added to the mastermix. Hybridization mixture for centromeric probes (1Opl):

Add to 1,5 ml vial. Mastermix for centromeric probes:

A

formamide use fume hood

Add mix to 15ml vial. Vortex thoroughly, adjust to pH Z5 with 2M HCI and make up to a final volume of 9ml with distilled water. The mastermix can be aliquoted and stored at -20 "C. The vial in current use can be stored at 4 "C. Hybridization mixture for Oncor translocation probes (60pl):

Fluorescence lrnrnunophenotyping and lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)

Mastermix for Oncor translocation probes:

Add mix to 15ml vial. Vortex thoroughly, adjust to pH 7.5 with 2M HCI, make up to a final volume of 10 ml with distilled water.

4.3.2 Chemicals Avid in-AMCA Biotin-labelled centromeric probe

Jackson Oncor

Biotin-labelled goat anti-avidin antibody bcr-abl probe Cot-1 DNA Cy3-coniugated goat anti-mouse antibody Cy3-conjugated rabbit anti-goat antibody Cy3-coniugated donkey anti-rabbit antibody Cy3-conjugated rabbit anti-mouse antibody DABCO Deionized formamide Dextran sulphate DIG-labelled centromeric probe

Vector Oncor Gibco BRL Jackson Jackson Jackson Jackson Sigma Gibco BRL Oncor Oncor

FITC-coniugatedanti-digoxigenin (monoclonal) FITC-coniugateddonkey anti-mouse antibody Mouse anti-FITC antibody (monoclonal) ,,class-matched” mouse antibodies : Mouse IgG 1 Mouse IgG 2a Mouse IgG 2b Mouse IgM Normal mouse serum Rubber cement: Fixogum Silane-coated slides Sonicated salmon sperm DNA (10 mg/ml)

Boehringer Jackson Dakopatts

Immunotech lmmunotech lmmunotech lmmunotech Jackson Marabu Camon Sigma

003-150-083 P5000-BioP5090-Bio BA-0300 5109-D/B 15279-011 115-165-068 305-165-045 701- 165- 152 315-165-048 D-2522 15515-018

54030 P5000-DGP5090-DG 1207741 715-095-151

M878 0571 0572 1266 1268 015-000-001 2901 10000

k0103 D9156

4.3.3 Equipment Standard equipment is used throughout, which can be obtained from many different commercial suppliers. Digital imaging of results was carried out on an lSlS imaging system supplied by Metasystems Hard and Software GmbH.

K. WEBER-MATTHIESEN

4.4 Preparation of Slides for FICTION Success with the FICTION technique is reliant upon the availability of high quality slides. The following samples can be used: cytospin slides, smears, imprints and cryostat sections. Users of the FICTION technique should familiarize themselves with the methods of preparing slides in specialized laboratories. Some general points should be noted. Freshly prepared slides should be air-dried overnight at room temperature (RT). Thereafter, slides can be processed immediately or stored airtight at -80 "C. Slides preserved in this way can be stored without deterioration for years; when single slides are taken out of the freezer, it is very important to make sure that the slides remaining in the freezer do not thaw. This is possible by taking the slides out of the container inside the freezer. If slides are stored at -20 "C, the quality of the immunophenotyping may be diminished after a few months; the quality of the hybridization is not affected. Before starting the FICTION procedure slides should be examined for the morphological quality of the cells by phase contrast microscopy. If many slides are available, only those with good cell morphology cell should be processed. If the cells are too densely packed, it is difficult to evaluate the immunophenotype of individual cells. Moreover, it may be hard to correlate the hybridization signals to individual nuclei. The number of cells per cytospin slide should not exceed 30,000 cells (diameter of the area containing cells = 0.5 cm). In imprints and smears, cells often overlap, but there are usually areas in the periphery where the cells lie separated from each other. Only such areas should be evaluated. The erythrocytes should lie flat on the slide and not be standing on end (Fig. 4). Sometimes, this is only possible at the end of the smears. Cryostat sections must be prepared on silane-coated slides [Maddox and Jenkins 19871, otherwise, the section will be lost during the hybridization procedure. Silane-coated slides are commercially available. Cytospin slides, smears and imprints do not require the use of silane-coated slides. It is recommended to hybridize the whole section. Partial hybridization of the section may result in the loss of the whole section.

preparation of slides

treatment of cryostat sections

Fluorescence lmmunophenotyping and lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)

Fig. 4. On hybridized smears only areas with good morphology should be evaluated. The erythrocytes should lie flat on the slide and not be standing on end

4.5 FICTION Protocol 1: lmmunophenotyping Using immuno phenotyping

One Antigen Plus In Situ Hybridization of Two Centromeric Probes 1. Fix slides in fresh acetone for 10 minutes at RT. Air-dry the slides for 10 min at RT. Cryo-preserved slides should be air-dried prior to fixation for at least one hour after being taken out of the freezer. After fixation, air-dry the slides in order to allow the acetone to evaporate. At this point it is possible to interrupt the staining procedure and continue the next day.

2. Apply lOOpl of monoclonal mouse antibody in PNM buffer (against the antigen that is to be stained) to the area of the slide containing the cells or the section. Incubate for 30 min at RT. The optimum dilution of the employed monoclonal antibodies must be determined by serial dilution. Many antibodies work well at a dilution of 1 :loo. After each antibody incubation step (steps 2, 3, 4, 14, 15 and 16) wash the slides 3 times for 2 min each in PN buffer. Washing is carried in glass jars. Before applying the antibody solutions, carefully dry the areas around the celWsections with a paper towel. (Attention: d o not damage cryostat sections or cells). Each antibody incubation is performed for 30 minutes at room temperature. K. WEBER-MATTHIESEN

3. Incubate with Cy3-conjugated goat anti-mouse antibody (1:200 in PNM buffer). 4. Incubate with Cy3-conjugated rabbit anti-goat antibody (1:200 in PNM buffer). 5. Incubate with Cy3-conjugated donkey anti-rabbit antibody (1:100 in PNM buffer). The four-step immunostaining technique used in this protocol employs polyclonal antibodies conjugated to the strongly fluorescent dye Cy3. It results in very strong fluorescent staining. If strongly expressed antigens, such as CD3, have to be stained, it is possible to omit step 5 or even steps 4 and 5. At this point the success of the immunostaining can be monitored. Monitoring of immunophenotyping is always recommended if the processed slides have been stored for a long period or if the cells showed poor morphology prior to the staining procedure. For this purpose the slides are mounted in PN buffer (not in glycerol!) and examined under the fluorescence microscope. The fluorescence intensity is relatively low if the slides are mounted in PN buffer. However, mounting in glycerol prior to in situ hybridization may cause hybridization artifacts.

6. Fix slides in 1 % paraformaldehyde for 1 min at RT in a glass jar. Appropriate fixation is one of the most important steps of the FICTION procedure. Fixation in 1 % paraformaldehyde for 1 min has turned out to be optimal. With lower concentrations of paraformaldehyde and shorter fixation periods the immunostaining may be lost during the harsh hybridization procedure. More intensive fixation diminishes the hybridization results.

7. Wash in distilled water for 2 min at RT. 8. Dehydrate the slides in a series of 70 %, 85 % and 100% ethanol at RT, each for 2 min. Air-dry the slides for 10 min at RT. 9. Apply centromeric hybridization mixture (detailed in 4.3.1) to the

fixation

@

hybridization

area to be hybridized; add coverslip and seal with rubber cement. The quantity of hybridization mixture depends on the size of the area to be hybridized. For hybridization of cytospin slides, 1.5~1is enough when using 8-10 mm round coverslips. If air bubbles occur, the amount of hybridization mixture should be increased. The amount must also be increased if larger coverslips are required.

10. Place the slides at the bottom of a metal box and denature for 3 min in a waterbath at 75 "C.

11. Immediately transfer the hot metal box into a 37 "C incubator. For centromeric probes one hour of hybridization is sufficient. However, hybridizations over a weekend are also possible. This neither diminishes the immunostaining nor the quality of in situ hybridization. Fluorescence lrnrnunophenotyping and lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION]

12. After removing the rubber cement wash the slides for 3 x 2 min in 50% formamide, lxSSC at 45 "C. 13. Equilibrate slides in PN buffer for 2 min.

@

visualization

During the following detection procedure biotin- and digoxigenin-labelled probes are visualized simultaneously:

14. Incubate slides with a mixture of avidin-AMCA and FITCconjugated monoclonal anti-digoxigenin antibody at RT for 30 min. [both 1 :200 in PNM buffer] 15. Incubate with a mixture of biotinylated goat anti-avidin antibody and mouse anti-FITC antibody at RT for 30 min. [both 1 :200 in PNM buffer]

16. Incubate with a mixture of avidin-AMCA and FITC-coniugated donkey anti-mouse antibody at RT for 30 min. [both 1:200 in PNM buffer].

If necessary, the fluorescence intensity can be amplified by multiple repetitions of steps 14 and 15. 17. Mount the slides in antifade solution. Now the slides can be analyzed under a fluorescence microscope.

If triple-dye fluorescence filter sets are used all three fluorescent

alternative stain

colors can be evaluated simultaneously. Alternatively, specific filter sets for AMCA, FlTC and Cy3 can be used to evaluate the different fluorescent colors separately. Appropriate filter sets are available from microscope-manufacturers and from filter-manufactures (e.9. Omega Optical). Note: Two color in situ hybridization is also possible using pairs of biotin and FlTC labelled probes [Weber-Matthiesen et al. 1993dl

4.51 Control Studies: Specificity of lmmunophenotyping

control specifity of immuno phenotyping

alternative approaches to ensure specifity K. WEBER-MAllHIESEN

Some cell types, for example granulocytes, carry ,,Fc receptors", on their membranes, which bind to the so-called ,FC fragment" of antibodies. Therefore, the Fc-fragments of polyclonal secondary antibodies (e.g. the Cy3-conjugated antibodies employed in the FICTIONprotocols) may bind to the Fc-receptors of human granulocytes. This type of cross reactivity may give the false impression of a positive reaction in granulocytes (Fig. 5). Two different approaches may be employed to negate this effect. One possibility is the use of so-called F(ab')2fragments, i.e. antibodies lacking the Fc-portion. The disadvantage of this approach is that F(ab'), fragments reveal a reduced fluorescence intensity. Therefore, the second approach is often more suitable. Whole molecule antibodies, i.e. those containing the Fc fragment, should be employed, which were selected by the manufacturers for minimal

Fereceptor binding unspecific

antigen binding specific

Fc receptor antigen

Cy3 conjugated secondary antibody

monoclonal antibody

Fig. 5. Mechanisms of non-specific Fc-receptor binding and specific antigen binding

crossreactivity with human tissue. All antibodies recommended in this chapter were selected for their minimal crossreactivity. As a rule, these antibodies do not bind to human Fc receptors. Nevertheless, specific antibody binding should be verified by the following test: One slide of each case is stained according to FICTION-protocol 1 with the following modification: the monoclonal mouse antibody is replaced with so-called ,,class-matchedH mouse antibodies, which must be non-reactive with human tissue. Class matched mouse antibodies have the same immunoglobulin subtype type as the monoclonal antibody they replace, e.g. IgG1, IgG2a, or IgM. With this approach, all cells on the slide should be unstained. Positive staining

con+rolslide

Fluorescence lrnmunophenotyping and lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)

on control slides processed in this manner always indicates nonspecific binding. A slight granular staining of all cells indicates that the cells are damaged. In this case, other slides should be stained.

4.6 FICTION Protocol 2: lmmunophenotyping (Two Different Antigens) Plus In Situ Hybridizat ion (0ne Centromeric Probe) Sometimes it is necessary to demonstrate two different antigens in one aberrant cell simultaneously. This is the case, for example, in chronic lymphocytic leukemia of B-cell type (B-CLL).In B-CLL, the leukemic cells are positive for both CD5 and CD19. In normal blood, Bcells express only CD19, normal T-cells only CD5. In consequence, immunophenotyping for CD5 or CD19 alone is not sufficient to differentiate single positive normal lymphocytes from double positive BCLL cells. Normal lymphocytes and B-CLL tumor cells can easily be distinguished by the simultaneous demonstration of both antigens. The technique of fluorescence double immunophenotyping is commonly used in FACS-analyses. However, the staining procedures for FACS are always one-step techniques using monoclonal antibodies which are directly labelled with different fluorescent dyes. Unfortunately, the fluorescence intensity of directly labelled antibodies is too weak to be evaluated under the fluorescence microscope. In order to reach sufficient fluorescence intensity, a multi-step procedure is necessary using polyclonal secondary antibodies as employed in FICTION-protocol 1. For this purpose, antibody cascades have to be established allowing the selective detection of the two antigens to be analyzed. The problem is, that the majority of available monoclonal antibodies are raised in the mouse. Thus, differently labelled secondary anti-mouse antibodies will always bind to both monoclonal antibodies. As a result, double positivity always appears to exist, even if only one antigen is expressed. The FICTION-protocol 2, which was specifically designed for fluorescence double immunophenotyping, allows two different antigens to be stained specifically with high fluorescence intensity (Fig. 6) [Weber-Matthiesen et al. 1993bl. Pretreat the slides as described in step 1 of FICTION protocol 1 (see 4.5) first irnrnuno phenotyping

1. Incubate with monoclonal antibody against antigen A (e.g. CD5) for 30 min at RT. Wash 3 x 2 min in PN buffer.

2. Incubate with Cy3-conjugated rabbit anti-mouse antibody (1 :200 in PNM buffer) for 30 min at RT. Wash 3 x 2 min in PN buffer. 3. Incubate with Cy3-conjugated donkey anti-rabbit antibody (1 :200 in PNM buffer) for 30 min at RT. Wash 3 x 2 min in PN buffer. 4. Incubate with 20% normal mouse serum diluted in PNM buffer for 15 min at RT. K WEBER-MATrHIESEN

3

avidinAMC4

Cy3 conjugated secondary antibody

A

normal mouse serum

bio inylated and-avidin antibody

Fig. 6. Principle of specific double immunophenotyping [indirect technique) according to FICTION-protocol 2

By incubating with normal mouse serum containing high concentrations of mouse immunoglobulins, the free binding sites of the Cy3-conjugated rabbit anti-mouse antibody (step 2) are blocked. This prevents the monoclonal antibody against antigen B, which is applied in step 5, from being attracted by the free binding sites of the rabbit antibody. Fluorescence lmmunophenoiypingand lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)

@

second immuno phenotyping

5. Incubate with biotinylated monoclonal antibody against antigen B (e.9. CD19) in PNM buffer containing 20% normal mouse serum for 30 min at RT. Wash 3 x 2 min in PN buffer. The optimum dilution for the biotinylated antibody must be determined by serial dilutions. As a rule, biotinylated monoclonal antibodies must be applied at higher concentrations than unlabelled monoclonal antibodies. The following detection procedure for antigen B is very similar to that used for the detection of biotinylated DNA probes:

6 .Incubate with AMCA-coniugated avidin (1 :200 in PNM buffer) for 30 min at RT. Wash 3 x 2 min in PN buffer. 7. Incubate with biotin-conjugated goat anti-avidin antibody (1 :200 in PNM buffer) for 30 min at RT. Wash 3 x 2 min in PN buffer.

8. Incubate with AMCA-coniugated avidin (1 :200 in PNM buffer) for 30 min at RT. Wash 3 x 2 min in PN buffer. AMCA-conjugated avidin and biotinylated anti-avidin antibodies used to visualize antigen B do not crossreact with the antibody cascade used to stain antigen A. Multiple amplifications of the AMCA staining are possible by repeating steps 7 and 8. The results of immunophenotyping can be monitored as described in FICTION-protocol 1, step 5 (see 4.5).

9. Fix in 1 % paraformaldehyde for 1 min at RT. 10. Dehydrate the slides in a series of 70%, 85% and 100% ethanol at RT for 2 min each. Air-dry the slides for 10 min at RT. At present only three suitable fluorescence colors are available. Therefore, only one DNA probe can be applied if double immunostaining is desired. In this protocol a digoxigenin labelled centromerespecific probe is applied. Attention: Do not use biotin-labelled probes, because they bind non-specifically to the antibody cascade of antigen B.

11. Apply hybridization mixture to the slide, cover with a coverslip and seal with rubber cement.

hybridization

K. WEBER-MATrHIESEN

The hybridization mixture is prepared in the same way as the "Hybridization mixture for centromeric probes" with one modification: 1PI of digoxigenin labelled probe (instead of 0.5~1 biotin labelled probe digoxigenin labelled probe) is added to 9pI of mastermix. The plus 0.5~1 quantity of hybridization mixture and the size of the coverslip depend on the area to be hybridized (see FICTION-protocol 1, step 9).

12. Place the slides at the bottom of a metal box and denature for 3 min at 75 "C in a waterbath.

13. Immediately transfer the hot metal box into a 37 "C incubator. Centromeric probes should be hybridized for at least 1 hour. 14. Wash slides for 3 x 2 min in 50 % formamide, 1xSSC at 45 "C.

15. Equilibrate in PN buffer for 2 min. 16. Incubate with a FITC-conjugated monoclonal anti-digoxigenin antibody at RT for 30 min (1:200 in PN buffer). Wash 3 x 2 min in PN buffer.

1Z Incubate with FITC-conjugated donkey anti-mouse antibody at RT for 30 min (1 :200 in PNM buffer). Wash 3 x 2 min in PN buffer. If required the fluorescence intensity can be amplified by repeating steps 18 and 19.

18. incubate with monoclonal mouse anti-FITC antibody for 30 min at RT (1 :200 in PNM buffer). Wash 3 x 2 min in PN buffer. 19. Incubate with FITC-conjugated donkey anti-mouse antibody for 30 min at RT (1 :200 in PNM buffer). Wash 3 x 2 min in PN buffer. Using a triple-dye filter set for the simultaneous evaluation of all the three fluorescent colors, negative cells, single positive cells and double positive cells can be easily distinguished. Fig. 6a and b were produced by sequentially digitalizing the different colors and phase contrast.

4.6.1 Control Studies: Specificity of the lmmunophenotyping Although the antibodies used for detecting the two different antigens do not cross react with each other, the specificity of immunophenotyping should be verified. This is possible as follows: monoclonal antibody against antigen B is replaced with biotinylated class-matched mouse antibodies non-reactive with human tissue. All other incubations are performed as described in FICTION-protocol 2. After this staining procedure only single positive Cy3 stained cells should be visible. If double positive cells are present, cross reactivity must be the reason. In our experience cross reactivity never occurs if 20 % normal mouse serum is used.

control specifity of double muno no phenotyping

Fluorescence lmmunophenotypingand lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)

4.7 FICTION Protocol 3: Detection of Structural Chromosome Aberrations Within lmmunophenotyped Cells The FICTION technique was originally designed for detecting numerical chromosome aberrations in immunophenotyped cells. Accordingly, the use of centromeric DNA probes is described in the FICTION-protocols 1 and 2. However, many neoplastic disorders are characterized by structural rather than numerical chromosome aberrations. Some of these structural aberrations are highly specific for certain disease entities or subtypes, and associated with the clinical course and the prognosis. Meanwhile, DNA probes have been developed, which allow structural chromosome aberrations to be detected in interphase cells by FISH [Arnoldus et al. 19901. Some of the new probes are already commercially available, for example probes for the Philadelphia-translocation t(9;22) (q34;q11), which is generally found in chronic myeloid leukemia (CML) and in some cases of acute leukemia. Employed in FISH-studies, these probes reveal a typical constellation of hybridization signals in Philadelphiapositive cells. The same probes can also be used in FICTION studies (Fig. 10).The following FICTION protocol 3 employs the bcr/abl translocation probe available from Oncor. Other Oncor probes for structural chromosome aberrations can be used in the same way.

1. Slides are immunostained as described in FICTION-protocol 1, steps 1-8 (see 4.5). 2. Apply hybridization mixture to the slide; cover with a coverslip and seal with rubber cement. For the preparation of hybridization mixture: see 4.3.1. The quantity of the applied hybridization mixture depends on the conditions described in FICTION-protocol 1, step 9.

3. Place slides to the bottom of a metal box and denature for 5 min at 75 "C in a waterbath. 4. Immediately transfer the metal box into a 37 "C incubator. Hybridize the slides overnight.

5. Wash 3 x 7 min in 0.1xSSC, pH Z5 at 60 "C

6. Detect the biotiddigoxigenin labelled probes is performed as described in FICTION protocol 1, steps 14-17. The bcr and abl probes show fluorescent hybridization signals which are much smaller than the signals of centromeric probes. The bcr genes are stained green (FITC),the abl genes are stained blue (AMCA).A certain constellation of the signals in the nucleus as shown in Fig. 10 indicates the presence of the Philadelphia translocation.

K. WEBER-MATTHIESEN

4.8 Evaluation of Results There are a few problems concerning the evaluation of the immunophenotype and the hybridization signals that need to be considered: Autofluorescence. Some cells show autofluorescence, which must not be confused with non-specific antibody binding; the latter must be avoided (see control studies). A typical feature of autofluorescence is that it is observed with all different filter sets: with the Cy3 filter it appears red, with the FlTC filter yellow, and with the AMCA filter white (not blue!).With a simple test one can clarify whether the red fluorescence is really owing to Cy3 immunostaining: True Cy3 fluorescence is not visible if the AMCA filter set is used. Moreover, the appearance of true Cy3 fluorescence is different from autofluorescence. Cy3 fluorescence is brilliant, while autofluorescence appears dull red. With some practice it is easy to distinguish true Cy3 fluorescence from autofluorescence. For the evaluation of AMCA and FlTC hybridization signals autofluorescence does not play a role. Evaluation of hybridization signals: it is difficult to evaluate the hybridization signals if the cells are located very close to each other. In such cases it is recommended to switch on the bright field lamp of the microscope in addition to the fluorescence lamp and to assign the hybridization signals to individual nuclei with the help of phase contrast (Fig. 7a, b; 8a, b). Evaluation of immunophenotype: if the cells are too close together the evaluation of the immunophenotype is also difficult. In particular, differentiating immunophenotypically negative cells among a group of positive cells may sometimes be impossible. In this case, areas on the slide should be analyzed where the cells are far enough apart. Variation in thickness of cryostat sections (Fig. 9): a problem with cryostat sections is that they vary in thickness from one area to the other. In thick areas the nuclei may overlap each other, while in thin areas the nuclei are often sliced, i.e. incomplete. For this reasons the evaluation of cryostat sections is generally difficult. This problem has been discussed elsewhere [Weber-Matthiesen et al. 1993~1. Rate of hybridization : the rate of successful hybridizations is lower on cryostat sections than on other samples. A relatively frequent finding is that instead of the expected hybridization signals only a diffuse staining of the whole nuclei is visible. This is most likely due to degradation of the DNA in the sliced nuclei. In our experience best hybridization results are obtained if the sections are prepared immediately after shock freezing of the biopsy specimen. FICTION should be performed right away after the freshly cut sections have been airdried.

how to distinguish a Cyo-signal from autof luorescence

addition of phase contrast

use cryostat sections immediately

Fluorescence lmmunophenotyping and lnterphase Cytogenetics as a Tool for Investigation of Neoplasms [FICTION)

Fig. 7. a CD30-positive binucleate Reed-Sternberg cell with three blue (chromosome 7) and three green (chromosome8) signals per nucleus (one blue signal not in focus). b same cell as shown in Fig. 7a: using simultaneous phase contrast the signals can be assigned to individual nuclei. One green signal appears to be exactly at the border between the two nuclei (arrow).This signal belongs to the left nucleus as was determined by altering the focus of the microscope. Note that the hybridization signals of the surrounding cells are out of focus. This is a general problem if very large cells and small cells are analyzed si muItaneously

Fig. 8a, b. [Above) Double immunophenotypingfor CD19 and CD5 in a case of B- b CLL; in situ hybridization with a centromeric .probe for chromosome 12; simultaneous phase contrast demonstration. a Two CD19-positivecells (red fluorescence) with green signals for chromosome 12 (some signals not in focus). b same cells as shown in Fig. 8a: Two cells are CD5-positive (blue fluorescence). One positive cell is partially damaged. Note that only one of the cells is positive for both CD5 and CD19 (large arrows in Fig. 8a and 8b). CD5/CD19 double positivity is a typical feature of B-CLL cells. Single positive cells in Fig. 8a and 8b are indicated by small arrows Fig. 9. (Below left) Cryostat section (reactive lymphoid tissue) processed according to FICTION protocol 1 using the B cell-specific anti-CD19 antibody and a centromeric probe for chromosome 8 (blue signals). The hybridization signals are clearly visible within the immunostained cells. Nuclei are demonstrated by simultaneous phase contrast. The FICTION method applied to cryostat sections allows the study of immunophenotypically characterised tumor cells with defined chromosome abberations within their natural context. Using this approach, the topography of tumor celVreactive cell interactions can be studied in situ

Fig. 10. (Below right) Philadelphia-positive, i.e. t(9;22)(q34;q11), acute myeloid leukemia (AML). This bone marrow smear was stained according to FICTIONprotocol 3 using an anti-CD13 antibody and DNA probes specific for the bcr gene (chromosome 22qll) and the abl gene (chromosome 9q34). The CD13-positive myeloid cell shows the signal constellation typical for the translocation t(9;22): Corresponding to the bcr/abl fusion a greedred double signal is visible (green = abl, red = bcr). Normal cells show four separate hybridization signals Fluorescence lmmunophenotyping and lnterphase Cytogenetics 0 s o Tool for Investigation of Neoplasms (FICTION)

4.9 Documentation of FICTION-Results documentation

(1) Documentation is possible by conventional photography. A 400 ASA film, for example Kodak Ektachrome 400, is recommended. If specific filter sets for Cy3, AMCA and FlTC are used, triple exposure is necessary. Exposure times lie in the range of 0.5 to 20 seconds. A further exposure with phase contrast bright field illumination is sometimes useful to show how signals relate to individual nuclei (Fig. 6-7). The quality of conventional photography is demonstrated elsewhere [Weber-Matthiesen et al. 1992, Weber-Matthiesen et al. 1993a-c, Weber-Matthiesen et al. 1995a-b] (2) Digital imaging is an easy alternative to conventional photographic documentation. All figures provided in this FICTION chapter were produced using the lSlS imaging system supplied by MetaSystems Hard & Software GmbH. Note: In most cases it is not possible to demonstrate all hybridization signals on one photograph because the signals are located in different focal planes of the nuclei. Some signals are regularly not in focus. This is particularly a problem if large cells, e.g. Hodgkin cells, have to be photographed.

\ troubleshooting

4.10 Troubleshooting Problem: lmmunophenotyping: No staining/high background staining. Possible reasons and solutions a) Inappropriate preservation of slides. Use freshly prepared slides for testing. Preserve slides according to 4.3. b) Monoclonal antibody is damaged. Test the antibody on freshly prepared slides obtained from different patients. c) One or several secondary antibodies are damaged. If unsatisfactory immunostaining occurs on different freshly prepared slides all antibodies have to be tested separately. Exchange all antibodies by new ones (only one per test). Problem: lmmunostaining is sufficient prior to ISH but less intensive after ISH. Possible reasons and solutions Paraformaldehyde is too old, replace. Problem: In situ hybridization: Too much background. Possible reasons and solutions a) Cross hybridization with other DNA regions. Increase the hybridization temperature. Prepare new wash solutions. b) Signal amplification too strong. Omit some amplification steps.

K. WEBER-MATTHIESEN

Problem: AMCA fluorescence intensity consistently too low. Possible reasons and solutions AMCA is very unstable. Buy new reagents and store in 50 % glycerol at -20 "C. Problem: No hybridization signals. Possible reasons and solutions a) Defective DNA probes. Test the probes on different freshly prepared slides. If unsuccessful, try a new probe. b) Paraformaldehyde solution is too old (pH!). Replace. c) Detection reagents are defective. Test : check the probe using another detection system, for example, the biotin instead of the DIG cascade, and vice versa.

References Arnoldus EPJ, Wiegant J, Noordermeer IA, Wessels JW, Beverstock GC, Grosveld GC, Van der Ploeg M, Raap AK (1990) Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet. Cell Genet. 54: 108-111. Baurmann H, Cherif D, Berger R. (1993) lnterphase cytogenetics by fluorescent in situ hybridization (FISH)for characterization of monosomy-7-associatedmyeloid disorders. Leukemia 7:384-391. Haas OA, Koller U, Ambros P, Kornmuller R, Maidic 0, Gadner H, Knapp W. (1987) lmmunoenzymaticstaining methods for simultaneous demonstration of chromosomes and cell surface markers. Cancer Genet. Cytogenet. 27:229-240. Knuutila S, Teerenhovi L. (1989) lmmunophenotyping of aneuploid cells. Cancer Genet. Cytogen. 41 : 1-17. Maddox PH, Jenkins D. (1987) 3-aminopropyltriethoxysilane (APES): a new advance in section adhesion. J. Clin. Pathol. 40:1256-1260. Pinkel D, Straume T, Gray JW. (1986) Cytogenetic analysis using quantitative, highsensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA. 83:2934-2938. Schlegelberger B, Noelle I, Feller AC, Bauer E, Grote W. (1990) Angioimmunoblastic lymphadenopathy with trisomy 3: the cells of the malignant clone are T cells. Hematol. Pathol. 4:179-183. Schlegelberger B, Weber-Matthiesen K, Eumann S, Rathjen M, Grote W. (1994) Detection of structural chromosome aberrations in immunophenotyped mitoses. Cytogenet. Cell Genet. in press:-. Schlegelberger B, Weber-Matthiesen K, Himmler A, Bartels H, Sonnen R, Kuse R, Feller AC, Grote W. (1994) Cytogenetic findings and results of combined immunophenotyping and karyotyping in Hodgkin's disease. Leukemia 8:72-80. Teerenhovi L, Knuutila S, Ekblom M, Rossi L, Borgstrom GH, Tallman JK, Andersson L, De la Chapelle A. (1984) A method for simultaneous study of karyotype, morphology and immunologic phenotype of mitotic cells in hematologic malignancies. Blood 64:1116-1122. Weber-Motthiesen K, Winkemann M, Muller-HermelinkA, Schlegelberger B, Grote W. (1992) Simultaneous fluorescence immunophenotyping and interphase cytogenetics: a contribution to the characterization of tumor cells. J. Histochem. Cytochem. 40:171-174 Weber-Matthiesen K, Deerberg J, Muller-Hermelink A, Schlegelberger B, Grote W. (1993a) Rapid immunophenotypic characterization of chromosomally aberrant cells by the new FICTION method. Cytogenet. Cell Genet. 63:123-125 Weber-Matthiesen K, Muller-Hermelink A, Deerberg J, Scherthan H, Schlegelberger B, Grote W. (1993b) Discrimination of distinct subpopulations within a tumor with combined double immunophenotyping and interphase cytogenetics. J. Histochem. Cytochem. 41 : 1641-1644 Fluorescence lmmunophenotyping and lnterphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION]

Weber-Matthiesen K, Press1 S, Schlegelberger 6, Grote W. (1993~)Combined immunophenotyping and interphase cytogenetics on cryostat sections by the new FICTION method. Leukemia 7: 646-649 Weber-Matthiesen K, Muller-Hermelink A, Winkemann M, Schlegelberger B, Grote W. (1993d) Rationalization of in situ hybridization : Testing up to 16 different probes on a single slide. Cancer Genet. Cytogenet. 68: 91-94. Weber-Matthiesen K, Deerberg J, Poetsch M, Grote W, Schlegelberger 6. (1995~) Clarification of dubious karyotypes in Hodgkin’s disease by simultaneous fluorescence immunophenotyping and interphase cytogenetics (FICTION). Cytogenet. Cell Genet. 70: 243-245. Weber-Matthiesen K, Winkemann M, Grote W, Schlegelberger 6. (199513) Kappa and lambda positive cells in centroblastic-centrocytic lymphoma (follicular lymphoma) may share the same secondary chromosome aberration: Reflections on early lymphoma development. Genes Chrom. Cancer 14: 22-27. Weber-Matthiesen K, Deerberg J, Poetsch M, Grote W, Schlegelberger 6. (1995~) Numerical chromosome aberrations are present within the CD30-positive Hodgkin and Reed-Sternberg cells in 100% of analyzed cases of Hodgkin’s disease. Blood 86: 1464-1468.

K. WEBER-MATTHIESEN

In Situ Hybridization Melody Clark Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 5

In Situ Hybridization to Embryo Whole Mounts and Tissue Sections: mRNA Detection and Application to Developmental Studies T. JOWETT, M. MANCERA, A. AMORES, and Y-L. YAN

5.1 Introduction An essential part of the molecular analysis of development is the determination of the spatial and temporal patterns of expression of a variety of different genes. This is most often performed initially at the level of detection of mRNA by in situ hybridization. Original methods employed radioactive probes but they have now been superseded by non-radioactively labelled antisense RNA in combination with immunohistochemical staining. Hybridization can be performed on tissue sections or on whole mounts of tissues or embryos. The temporal and spatial pattern of expression of a gene is most conveniently visualised in a single hybridization to a large number of whole embryos of different developmental stages. Once characterised such gene specific probes become useful cell markers for determining alterations to the normal pattern of events in embryos whose development has been perturbed by experiment or mutation. In situ hybridization has therefore become an important tool to those working with the major developmental systems: Drosophila, zebrafish, chick, Xenopus and mouse. It also provides a way of examining the molecular basis of development in other less experimentally amenable organisms. The process of in situ hybridization to whole embryos and to sections involves the following stages.

1. 2. 3. 4. 5. 6. 7.

Synthesis of labelled antisense RNA probe(s). Fixation and pretreatment of tissue. Hybridization of probe. Washing to remove unbound probe. Incubation in antibody against the probe-hapten. Washing unbound antibody. Visualisation of the bound antibody with immunohistochemical stain.

The method of in situ hybridization was first developed in Drosophila but can now be applied to a wide variety of biological material. However, in applying the technique to a particular organism there may be minor modifications required in order to obtain the optimum signal with the least background. M. Clark (Ed.), In Situ Hybridization. 0 Chapman & Hall, Weinheim. ISBN 3-8261-0038-7

It is usually necessary to relate the pattern of expression of a novel gene to that of other genes which are expressed at similar developmental stages and possibly in overlapping domains. This is essential in proposing models of regulatory gene networks controlling development. Comparisons between different gene transcripts can be performed by probing serial sections on separate slides with different probes but this is labour intensive and time consuming. Alternatively, two probes can be differentially labelled allowing simultaneous localisation of their corresponding target mRNAs in either whole embryos or tissue sections. We describe here two methods by which this can be achieved, described diagrammatically in Figures 1A and 1 B. Both methods can be applied to either whole mounts or sections and we present protocols for both procedures.

5.2 Materials and Chemicals 5.2.1 Buffers and Reagents BClP (5-bromo-4-chloro-3-indolyl-phosphate also known as X-phosphate): dissolve at 50 mg/ml in dimethylformamide. Store in aliquots at -20 "C. lox Digoxigenin nucleotide labelling mix: 10 mM each ATP, GTP, CTP; 6.5 mM UTP; 3.5 mM digoxigenin-UTP Store in aliquots at -20 "C.

ELFTM-APsubstrate kit (Molecular Probes): dilute the substrate 1 :20 in ELFTM Reaction Buffer supplied with the kit. Use within 30 min. Tissues should be equilibrated with pre-reactionwash buffer (30 mM Tris, 150 mM NaCl pH 7.5) prior to adding the diluted substrate solution. The reaction is stopped with 25 mM EDTA, 0.05% Triton X100 in PBS, the final pH should be 7.2 (addition of 1.0 mM levamisole is optional). Fast Red tablets, alkaline phosphatase substrate (Boehringer): each tablet contains 0.5 mg naphthol substrate, 2 mg Fast Red chromogen, and 0.4 mg levamisole. Store tablets for a short term at 4 "C or at -20 "C for long term. Wear gloves and use plastic forceps to handle the tablets. Dissolve one tablet in 2 ml of 100 mM Tris-HCI pH 8.2. Use the solution within 30 min.

lox Fluorescein nucleotide labelling mix: 10 mM each ATP, GTP, CTP; 6.5 mM UTP; 3.5 mM fluorescein-UTP Store in aliquots at -20 "C. Formaldehyde fixative: 4 % formaldehyde/PBS + 2 mM EGTA. Made directly by diluting 1 vol. 40% formaldehyde with 9 vol. PBS. Add 1M NaOH to pH Z5 and EGTA to 2 mM. Glutaraldehyde: 50% glutaraldehyde for EM (BDH) stock solution of glutaraldehyde is stored in aliquots at -20 "C.

MABT: 100 mM maleic acid; 150 mM NaCI, 0.1 % Tween-20, pH Z5. MABTB: MABT + 2 % blocking powder. Make a 10 % stock solution by heating 1g of Blocking powder (Boehringer)in 10 ml of MABT. Dissolve, autoclave and store in aliquots at -20 "C.

T. JOWETT et al.

A. Alkaline phosphatase and horseradishperoxidasestaining mRWml)m

1. Hybridise both ribopmbes 2. Incubate with both antibodies

sheep anti-digoxigenin-

3. Stain with diaminobenzidine

alkalinephosphatase

4. Stain with AP substrate

DAB+H,02

brow

Fast Red or NBTlBCIP

red or bluelpurple

B. Sequential alkaline phosphatase staining mRWmnthmn

1. Hybridise both ribopmbes 2. incubate with first antibody

3. Stain with NBT/BQP 4. Inactivate alkaline phosphahse

alkaline phasphatase

dkdinephosphatase

5. Incubate with second antibody

NBTlBClP

6. Stain with Fast Red

Fast Red

bludpurpk

red

C. Enzyme labelled fluorescence alkaline phosphataeo detection mRIUImlIsmw

1. Hybridise ribopmbe

2. Incubate with antibody 3. Incubate with ELF substrate 4. Irradiate with W

sheep anti-digmigeninalkaline phosphatase

5. View fluorescencewith DAPI filter set EnLabelled Flwreecence substrate

coloudess product

n yellowlgreen

uv

llght

fluorescence

Fig. 1 A-C. Strategies for performing non-isotopic in situ hybridization. (A) A mixture of antisense riboprobes labelled with digoxigenin and fluorescein are hybridised with the target mRNAs in the tissue. Following hybridization the tissues are incubated in an antibody mixture of anti-digoxigenin-horseradish peroxidase (POD) and anti-fluorescein-alkaline phosphatase (AP). The former is visualised with diaminobenzidine (DAB) and the latter with either nitrobluetetrazolium (NBT/ BCIP) or Fast Red. This gives a brown precipitate for the digoxigenin-labelled probe and a blue or red one for the fluorescein-labelled probe. (B) A mixture of antisense riboprobes labelled with digoxigenin and fluorescein are hybridised with the target mRNAs in the tissue. Following hybridization the tissues are incubated in a single antibody, anti-digoxigenin-alkaline phosphatase, and the signal visualised with NBT/BCIP The alkaline phosphatase is then inactivated by heating or treating with 100 mM glycine pH 2.2, before incubating in the second antibody, antifluorescein-alkaline phosphatase.The second signal is visualised by using Fast Red (Boehringer)as a substrate for the alkaline phosphatase which gives a red precipitate. (C) A fluorescent signal can be seen using the ELFTM alkaline phosphatase system. After hybridization and incubation with the alkaline-phosphatase coniugated antibody, the tissue is incubated in a solution of the ELFTM substrate. This is converted into a compound which will fluoresce when irradiated with UV light. The yellow/green fluorescent signal is visualised using a DAPI filter set

In Situ Hybridizationto Embryo Whole Mounts and Tissue Sections

mHybe: 50 % formamide, 5x SSC, 5 mM EDTA, 2 % Blocking powder, 0.1 % Tween20,0.5 % CHAPS, 50 pg/ml yeast RNA, 50 pg/ml heparin, pH to 4.5 with citric acid. Store in aliquots at -20 "C. Mouse embryo powder: Homogenise < - >12.5-14.5d mouse embryos in minimum volume of PBS. Add 4 volumes of ice-cold acetone, mix and incubate on ice for 30 min. Spin at 10,000~g for 10 min and remove supernatant. Wash pellet with ice-cold acetone and spin again. Spread the pellet out and grind it into a fine powder on a sheet of filter paper and allow it to air-dry. Store in an air-tight tube at 4 "C. NBT (4-nitro blue tetrazolium chloride): dissolve at 75 mg/ml in 70% dimethylformamide. Store in aliquots at -20 "C. NTMT: 100 mM NaCI, 100 mM Tris-HCI pH 9.5, 50 mM MgC12, 0.1 % Tween-20. Make from concentrated stock solutions on day of use (the pH will decrease on storage, due to absorption of carbon dioxide). Paraformaldehyde/PBS fixative: paraformaldehyde is dissolved in PBS at 65 "C. If it does not readily dissolve add a drop or two of 1 M NaOH solution to pH 7.5. It should be cooled to 4 "C and used within two days.

PBS (Phosphate Buffered Saline): 130 mM NaCI; 7 mM Na2HP04.2H20;3 mM NaH2P04.2H20.For a l o x PBS mix 75.97 g NaCI; 12.46 g Na2HPO4.2H,O; 4.80 g NaH2P04.2H20.Dissolve in less than 1 litre of distilled water; adjust to pH 7.0 and to final volume of 1 litre, sterilise by autoclaving. PBT: PBS, 0.1 % Tween-20. Proteinase K: make up a stock solution of proteinase K at 20 mg/ml in 50% glycerol 10 mM Tris pH 7.8 and store at -20 "C. sHybe: 50 % formamide, 1 . 3 ~ SSC, 5 mM EDTA, 0.2 % Tween-20,0.5 % CHAPS, 50 pg/ml yeast RNA, 100 pg/ml heparin, pH to 5.0 with citric acid.

20x SSC for hybridization and washing: (20x SSC is 3 M NaCI, 300 mM tri-sodium citrate). Dissolve 175.3 g NaCl and 88.2 g sodium citrate in 800 ml water. Adjust the pH with 1M citric acid to 4.5,5 or 6.0 depending on the hybridization solution used. Adjust the volume to 1 L and sterilise by autoclaving. 10xTBST: For 100 ml mix the following: NaCI, 8.0 g; KCI, 0.2 g; 1M Tris-HCI pH 7.5, 25 ml; 10% Tween-20, 10 ml. Dilute to l x and add levamisole to 2 mM on day of use.

lox Transcription buffer: 400 mM Tris-HCI pH 8.0; 60 mM MgCI,; 100 mM dithiothreitol; 20 mM spermidine; 100 mM NaCI; RNase inhibitor 1 unit/$. Store in aliquots at -20 "C. VectorTMRed alkaline phosphatase substrate (Vector Labs). Mix stock solutions as described with kit. Zebrafish acetone powder. Grind 4 g of adult fish in liquid nitrogen in a precooled mortar and pestle. Add 16 ml of 0.85% saline and then 16 ml of -20 "C acetone. Mix vigorously and keep at 4 "C for 30 min. Collect the precipitate by centrifugation at 10,000~g for 10 min. Resuspend in fresh cold acetone and allow to sit at 4 "C for 10 min. Respin at 10,000~g for 10 min. Transfer to clean filter paper (Whatman Grade 50 Hard) on aluminium foil. Allow to dry, spreading and dispersing until dry. Grind again in mortar and pestle. Collect powder.Transfer to airtight container. The yield is 10-20% of the original wet weight. T. JOWETT et al.

zHybe: 50% formamide, 5x SSC, 01 % Tween-20,500 p.g/ml yeast RNA, 50 p.g/ml heparin, pH to 6.0 with citric acid.

5.2.2 Chemicals Reagent

Supplier

alkaline phosphatase red staining kit anti-digoxigenin alkaline phosphatase (AP) Fab anti-digoxigenin horseradish peroxidase (POD) Fab anti-fluorescein alkaline phosphatase (AP) Fab Blocking Powder BSA albumen bovine fraction V CHAPS diaminobenzidine digoxigenin-11-UTP dimethylformamide DMSO DNasel, RNase free DPX mountant DTT (dithiothreitol) ELFTM-alkaline phosphatase detection kit

Vector Labs Boehringer Boehringer

Fast Red tablets fluorescein-12-UTP formamide glutaraldehyde 50% for EM heparin Histo-ClearTM levamisole maleic acid nitrobluetetrazolium (NBT) paraffin wax, Polywax paraformaldehyde proteinase K ribonucleoside triphosphate set RNA from Baker’s yeast RNA polymerase SP6 RNA polymerase T3 RNA polymerase T7 RNase A RNase inhibitor SDS serum sheep spermidine trihydrochloride TESPA tetrahydronaphthalene Tween-20 X-phosphate 4-toluidine salt (BCIP) ~~~

Boehringer Boehringer BDH Sigma Sigma Boehringer Aldrich BDH Boehringer BDH Boehringer Molecular Probes Boehringer Boehringer Prolabo BDH Sigma National Diagnostics Sigma Sigma Boehringer Difco Sigma Boehringer Boehringer Sigma Boehringer Boehringer Boehringer Pharmacia Boehringer BDH Gibco BRL Sigma BDH Aldrich Sigma Boehringer

Cat No. SK-5100 1093274 1207733

1426338 1096176 44155 C 3023 D5637 1209256 D 15,855-0 28216 776785 36029 708984 E-6601 1496 549 1427857 24311.291 36218 H7005 HS-200

L 9756 M0375 1087479 8829-19 P 6148 1000 144 1277057 R6750 810274 1031163 881767 27-0323-01 799017 44215 2V 035-6070H S 2501 66023 10,241 -5 P-1379 760994

5.2.3 Equipment The following equipment and consumables are available from histological suppliers. In Situ Hybridization to Embryo Whole Mounts ond Tissue Sections

Coplin jars (glass), Stretton Young jars (polypropylene), coverslips of different sizes, diamond writer, disposable microtome blades, Dual Hybridization Oven (Hybaid), glass embryo dishes, glass slide racks, glass staining jars, plain and frosted microscope slides, plastic embedding cassettes, rotary microtome, orbital shaker or rocker, slide mailers, slide warming tray, square Petri dishes, stainless steel embedding moulds, stainless steel slide racks.

5.3 General Procedures 5.3.1 RNase-free Treatments

how to avoid RNase contaminations

Reasonable care should be taken to prevent contamination with RNases. Most care should be taken in synthesis of the probe. A single synthesis should be enough for 100-200 hybridizations so it is worth storing it in aliquots at -20 "C in the presence of RNase inhibitor. Alternatively, it can be stored in hybridization solution which would make it completely resistant to RNase attack. Tissue will normally contain endogenous RNases which cannot normally be eliminated so rapid fixation is the best way of avoiding potential problems. Solutions are usually made with sterile deionised water and if possible autoclaved prior to use. We only use diethyl pyrocarbonate (DEPC)-treatedwater for synthesis and storage of the probe. Glass vials and microfuge tube used for fixing and subsequent storage of embryos may be autoclaved but this is usually not necessary. All solutions used prior to prehybridization are made from sterile stock solutions with deionised water in 50 ml screw-capped, polypropylene, centrifuge tubes (Falcon or similar). If these are made up fresh, they should be free from RNase contamination.

5.3.2 RNA Probe Synthesis 1. This is essentially according to the method recommended by Boehringer. Linearised plasmid equivalent to 1 pg of insert DNA is digested with proteinase K (0.05 pg/pI) for 30 min at 37 "C synthesis of RNA-probe

T. JOWETT eta/.

prior to phenokhloroform extraction and ethanol precipitation. We treat with proteinase K to ensure that the DNA is free of RNase prior to riboprobe synthesis. The DNA is redissolved in TE (10 mM Tris HCI, 1 m M EDTA pH 8.0) made with RNase-free water so that 1 pg of insert DNA is in 4 pI.

2. Mix together

Incubate the mixture for 2 hour at 37 "C.

3. Add 40 units of DNase I and incubate at 37 "C for 15 min to remove the plasmid DNA. Stop the reaction by adding 2 pl of 200 mM EDTA pH 8.0 and precipitate the RNA with 2.5 pI 4 M LiCI, and 75 pI prechilled ethanol. Spin down the pellet and redissolve in 100 pI of RNase-free water containing 40 units of RNase inhibitor. The probe can be checked by running 2.5 pl on an agarose minigel. Wash the apparatus thoroughly before preparing the gel and run the samples quickly to avoid problems with RNase. LiCVethanol precipitation does not completely remove unincorporated rNTPs but this is not normally a problem. An alternative would be to use a Microcon column (Anachem). In the case of fluorescein probes the pellet is yellow and unincorporated fluorescein-UTP runs at the front during electrophoresis and is easily seen on the UV-transilluminator. Probes are stored in aliquots at -20 "C. Previous protocols required that the probe should be partially hydrolysed by heating in a sodium bicarbonate buffer (Wilkinson, 1992).This generates shorter lengths of probe which more easily penetrate the tissue. However, we find that this hydrolysis is difficult to control and tends to give lower signals and now many laboratories omit this step. We have had success with full length probes ranging from 0.3-3.0 kb. Perhaps with larger probes (>6 kb) partial hydrolysis may be worth considering.

analysis of probe

partial probe hydrolysis?

5.4 Whole Mount In Situ Hybridization 5.4.1 Fixation and Pretreatments Aldehyde fixatives are the most commonly used for both whole mounts and tissue sections. They cross-link the material retaining the RNA, but still allow penetration of the probe and antibodies. Three commonly used fixatives of increasing cross-linking ability are 4 % paraformaldehyde, 4 % formaldehyde and 1 YO glutaraldehyde

In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

preparation of large embryos

respectively, each in phosphate buffered saline (PBS). Tissues are soaked in fixative solution for different periods of time depending on the size of the embryos or tissue. Zebrafish embryos of any age and mouse embryos less than 9 days are sufficiently fixed in 2 hours at room temperature but can be conveniently left overnight at 4 "C. Mouse and chick embryos must also be carefully dissected away from the extra-embryonic membranes as these can trap reagents in subsequent steps leading to background problems. If there are any other cavities which may trap reagents then they should be opened. It may be necessary to dissect >10.5d mouse embryos into smaller pieces; for example, to cut transversely through the neural tube to avoid the trapping of reagents in the lumen.

1. Dissect embryos in PBS (or PBS + 2 m M EGTA to inhibit RNases). fixation and dehydration of embryos

2. Fix in 10 ml 4 % paraformaldehyde in PBS at 4 "C, 2h-overnight. Alternatively, use 4 % formaldehyde/PBS + 2 m M EGTA. 3. Wash twice in PBT (PBS, 0.1 % Tween-20) at 4 "C. 4. Dehydrate with a series of methanol/PBT solutions (25%:75%, 50%:50%, 75%:25% methanol:PBT), then twice with 100% methanol. Incubate 10 min in each solution. Note that we use methanol rather than ethanol as ethanol causes the phosphate in PBT to precipitate.

storage of embryos

how to avoid damaging of embryos

T. JOWETT et al.

The initial dissection is best performed in a sterile embryo dish and subsequently the embryos are transferred to a sterile glass vial with polythene lid (Squat sample vials BDH Cat No. 215/0074/21) for fixing and dehydration. Zebrafish embryos are usually fixed inside their chorions and then dechorionated prior to dehydration in methanol. However, if they are fixed and dehydrated without dechorionation a white precipitate forms in the liquid between the chorion and vitelline membrane. This precipitate can be washed away following dechorionation and appears not to interfere with subsequent in situ hybridization. Zebrafish embryos can be stored in methanol for 1-12 months without detrimental effect. Prolonged storage of mouse, chick and Xenopus embryos in this way can lead to higher background problems so these embryos are better dehydrated in methanol and processed through to prehybridization solution before storage at -20 "C. (See steps 5 to 7 in this section). After dehydration we transfer zebrafish embryos from the glass vials which were used for prolonged storage in methanol to 1.5 ml microfuge tubes. We routinely hybridise 1-100 embryos per tube. Embryos are easily damaged and so care must be taken when transferring them. A 1 ml blue disposable pipette tip which has had its end cut off with a scalpel is suitable for this purpose. Larger embryos such as chick, mouse and Xenopus are better treated in 6-10 ml round bottomed tubes for the major washes and 2 ml polypropylene freezing vials (Nunc) which have a round bottom for hybridizations and anti-

body incubations. The embryos settle to the bottom of the tube and solutions can be replaced by gentle pipetting. When changing sohtions never withdraw all the liquid from the embryos, otherwise they will be damaged. We always include the methanol dehydration step with zebrafish embryos as they are usually stored in methanol at least overnight before starting - the hybridization. The methanol treatment originates from the protocol for' Drosophila embryos which requires a heptane and methanol treatment to remove the vitelline membrane [Forbes and Ingham, 1993).We do not know if this step is absolutely necessary for vertebrate embryos, but it is likely to help permeabilise the tissues so we include it.

5. Rehydrate the embryos through a methanol/PBT series (75%:25%, 50%:50%, 25%:75% methanol: PBT) finishing with three washes of PBT. At this stage it is necessary to bleach Xenopus embryos with 6 % hydrogen peroxide in PBT for 1 hour. This bleaches the pigment granules which would otherwise obscure the signal. Alternatively, bleaching may be performed after ISH, staining and fixation (Harland, 1991). It also helps to reduce background but can reduce signal. So, unless pigmentation is a problem, it is best left out.

dehydration of

rehydration, permeabilisation, fixation and prehybridization of embryos

6. Treat with proteinase K (10 pg/ml in PBT) to increase the perme-

ability of the membrane. This is performed for 10-20 min at room temperature depending on the type of embryo and the stage of development. The time may also depend on the particular batch of enzyme. Earlier protocols stopped the proteinase K digestion by replacing the solution with 2 mg/ml glycine in PBT. This is unnecessary and for zebrafish embryos we refix with 4 % paraformaldehyde in PBT for 20 min at room temperature. Slightly stronger fixatives (4 % paraformaldehyde, 0.2 % glutaraldehyde in PBT or 4 % formaldehyde, 0.1 % glutaraldehyde in PBT) are recommended for mouse, chick and Xenopus embryos. This second fixation is essential to prevent further proteinase K digestion and to prevent the embryo from disintegrating.

z

After fixing wash twice for 5 min in PBT and then add 0.2-1.0 ml of hybridization buffer (see Table 1). The volume used depends on the number and size of embryos. After a short incubation (5 min) replace the hybridization solution and incubate at 60-70 "C for a minimum of 2 hours. We lay the tubes almost horizontal in an upturned empty yellow pipette tip rack. In this way the embryos spread out and do not stick together. We incubate our embryos in a Hybaid Dual Oven which has a shaking table and gently shake the tubes at the lowest speed. This is not essential for hybridization but does facilitate the washing. Once in hybridization solution the embryos can be stored at -20 "C. The embryos are now safe from RNases.

In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

5.4.2 Hybridization 1. After prehybridization replace the solution with preheated hybridization mix containing probe. We use probes at 0.5 yglml hybridization

in zebrafish hybridization mix (zHybe). Probes which give strong signals may be effective at 0.1 pg/mI. For mouse, chick and Xenopus it is worth using 1 pg/ml initially and if you have background problems trying less.

2. Hybridization to labelled probe is usually carried out overnight between 60 and 70 "C.

species-specif ic

optimization of hybridization conditions

T. JOWETT et al

The basic hybridization mix for zebrafish is 50% formamide, 5x SSC, 500 pg/ml yeast RNA, 50 pg/ml heparin, 0.1 % Tween 20 brought to pH 6.0 with 1M citric acid (zHybe) and most of our hybridizations are at 65 "C. If background is a problem for a particular probe, it is worth increasing the formamide concentration and hybridization temperature. Many probes will still give a satisfactory signal in 65 % formamide at 70 "C. We also provide two alternative hybridization conditions (mHybe and sHybe) which are used for mouse, chick and Xenopus embryos. The mHybe solution is of medium stringency and originally suggested by D. Wilkinson. It includes Blocking powder (Boehringer), EDTA and CHAPS (3-[(cholamidopropyl)-dimethylammoniol-1-propane-sulphonate), a non-denaturing detergent. The pH of the hybridization mix varies from 4.5 to 6.0. In optimising the zHybe solution we tried pH 4.5, 6.0 and 7.0 and obtained good signals at all three pH values. The embryos were more fragile at pH 4.5 although the signal was marginally stronger. The sHybe solution (Henrique et al., 1995) is of higher stringency because of its reduced salt concentration and high hybridization temperature. Recipes for three hybridization mixes used for vertebrate embryos are provided in Table 1. The recipe for zebrafish is derived from that for Drosophila embryos. The other two will probably also work on Drosophila, but we have not tried them.

Reagent (stock solution)

Zebraf ish zHybe

Medium stringency mHybe

Stringent buffer sHybe

formamide (100%) ssc (20x) EDTA (0.5M pH 8.0) yeast RNA (20 mg/ml) Tween-20 (20 %) CHAPS (10%) heparin (50 mg/ml) Blocking powder citric acid (1M) hybridization temperature

50-65 % 5x

50 % 5x 5 mM 50 pg/ml 0.1 % 0.5 % 50 pg/ml 2% to pH 4.5 55 "C

50 %

500 pg/ml 0.1 % 50 pg/ml to pH 6.0 60-70 "C

1.3~ 5 mM 50 pg/ml 0.2 %

0.5% 100 pg/ml to pH 5.0 70 "C

5.4.3 Post-Hybridization Washes After hybridization it is necessary to wash off unbound probe.This must be done at the temperature of hybridization with prewarmed solutions. The double stranded RNA formed during the hybridization is very stable with a melting temperature considerably higher than equivalent RNA/DNA or DNA/DNA hybrids which is the reason that such stringent hybridization conditions can be used. The double stranded RNA is resistant to RNase digestion. Earlier protocols incorporated an RNase digestion to eliminate unbound probe but now this step is omitted as it causes a reduction in signal. However, since some probe may be bound to related non-homologous sequences it is advisable to prove the specificity of the signal by at least on one occasion incorporating an RNase treatment post hybridization. (Two 30 min washes in 20 pg/ml RNase A in 500 mM NaCI, 10 mM Tris-HCI pH 7.5,O.l % Tween-20.) We use the following protocol for zebrafish embryos. Wash the embryos for 10 min in 50% formamide, 5x SSC at the hybridization temperature. Then three similar washes with the same solution mixed with 2x SSC in the ratios 3: 1 ; 2:2; and 1 :3. Finish with a wash in 2x SSC alone at the hybridization temperature. We then perform two stringent 30 min washes in 0 . 2 ~SSC at 70 "C. This is best done on the shaker in the hybridization oven. Alternatively, use a heating block or water bath but gently invert the tube every 5-10 min. The embryos can be quite sticky at this stage and so should be treated with care. This problem can be avoided by adding Tween20 to each of the wash solutions to a final concentration of 0.01 %. Wash the embryos in the following mixes of 0 . 2 ~ SSC/PBT: 3: 1 ; 2:2; 1 :3 and finally in PBT. The initial washes are with 0.5 ml of solution while those in 2x SSC and later are 1 ml. The washes for larger embryos such as mouse, chick and Xenopus should be longer and use a larger volume (5-10 ml) of wash solution. Better results are obtained using maleic acid based buffers (Lamb et al, 1993). First rinse the embryos twice in prewarmed hybridization mix and then wash twice for 30 min in the same mix. Wash 20 min at hybridization temperature in prewarmed 1 : 1 hybe mix: maleic acid buffer (MABT: 100 m M maleic acid; 150 m M NaCI, 0.1 % Tween-20 pH 7.5). Rinse three times followed by two 30 min washes with MABT at the hybridization temperature.

RNase treatment

@

removal of unbound probe

5.4.4 Incubation with Antibody It is usual to block non-specific binding sites prior to applying the antibody. For zebrafish embryos we replace the PBT with PBT containing 5 % sheep serum, 2 mg/ml BSA, 1 % DMSO and incubate at room temperature for 1 hour on an orbital shaker. We then replace the solution with a similar mixture containing the antibody to the hapten used on the probe.

@

antibody incubation

In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

antibody dilution

choice of hapten

The titre of antibody used depends on the hapten and the nature of the visualisation step as well as the incubation time. (See Table 2.) A good starting point is 1 :2000 for anti-digoxigenin alkaline phosphatase if using NBT/BCIP as the visualisation step. Titres as low as 1 :8000 will also work if the antibody incubation is overnight at 4 "C. For the anti-digoxigenin horseradish peroxidase use 1 :1000. We find little difference in the sensitivity of anti-digoxigenin and anti-fluorescein Fab fragments conjugated with alkaline phosphatase (Boehringer)at the titres we use although other workers suggest that the anti-fluorescein Fab fragments are less sensitive (Strahle and Ingham, 1994). Direct comparisons are difficult because signal also depends on the level of incorporation of the hapten into the antisense probe. For mouse, chick and Xenopus replace the MABT with MABT + 2 % Boehringer Blocking powder (MABTB).Wash for 1 hour at room temperature. Replace with MABTB + 20% sheep serum and incubate for a further 1-2 hours. (The sheep serum should be heat-treated at 56 "C for 30 min). Replace with fresh solution containing the antibody and incubate overnight at 4 "C or 4 hours at room temperature.

5.4.5 Preabsorption of Antibodies

reduction of background signal

@ zebrafish acetone powder

@ mouse acetone powder

T. JOWEll et al.

It is advisable to preabsorb the antibody against either the type of embryo to be probed or an acetone powder made from the same organism. For zebrafish we make an acetone powder from adult fish whereas for mouse, chick or Xenopus we prepare equivalent powders from embryos. (See Section 5.2.1). Preabsorb the antibody with the appropriate acetone powder in blocking solution for 1-2 hour at room temperature. We use zebrafish powder at 1 % i.e. 1 mg/100 PI. (See Table 2 for suggested dilutions.) For preabsorption add 1 /400 dilution of antibody in 5% sheep serum, 2 mg/ml BSA, 1 % DMSO in PBT. Incubate' on a shaker for 60 min. at-room temperature. Spin at 10,000~ q for 10 min. Use the supernatant at the appropriate dilution. [See .. . Table 2 for suggested dihtions.) For mouse acetone powder add 3 mg of powder to 0.5 ml TBST (140 mM NaCI, 27 mM KCI, 250 m M Tris-HCI pH Z5,O.l % Tween20) with 5 PI sheep serum and 1 pI (0.75U) anti-digoxigenin-alkaline phosphatase or anti-fluorescein-alkaline phosphatase antibody. Incubate on shaker at room temperature for 1-2 hours. Spin in microfuge for 5 min and take off the supernatant into a new tube. Dilute with block solution to the working concentration.

Table 2. Antibody concentrations for whole mount in situ hybridizations Antibody

Stock

Working titre

Dilution

anti-digoxigenin-alkaline phosphatase, Fab fragments

150 U / 200 pI

0.37 U / ml

1 :2000

anti-digoxigenin-horseradish peroxidase, Fab fragments

150 U lyophilised 0.75 U / ml

1 :200

anti-fluorescein- alkaline phosphatase, Fab fragments

150 U / 200 pI

1 :2000

0.37 U / ml

5.4.6 Post-Antibody Washes and Histochemistry All washes are performed by laying the tubes on their side and gentle shaking on an orbital shaker. Some embryos contain endogenous alkaline phosphatase which will give background problems. This should be checked for by performing a control in which no antibody is added. Often endogenous activity is destroyed by the hybridization conditions or by pretreatment with hydrogen peroxide. However, if some endogenous activity persists include 2 mM levamisole in the NTMT. Only include levamisole if absolutely necessary as it decreases the signal. We have not found this to be needed in zebrafish, mouse or chick but it must be included in experiments on Xenopus embryos.

check for endogenous enzyme activity

1. Wash 6x 10 min with PBT containing 0.1 % BSA.

2. Wash twice with PBT for 30 min.

3. Wash three times for 10 min with NTMT (100 mM NaCI, 100 mM Tris-HCI pH 9.5, 50 mM MgCI2). Prior to the staining reaction for alkaline phosphatase it is important to equilibrate the tissue with buffer of pH 9.5. NTMT is buffered weakly with Tris and so with prolonged storage may develop a lower pH by absorbing carbon dioxide. Therefore make the buffer freshly just prior to use.

visualization of AP signal

make NTMT fresh

4. Incubate with NTMT including 4.5 PI NBT, 3.5 PI BClP per ml. Rock for the first 20 min. Transfer the solution and embryos to a glass embryo dish or glass vial for easier observation (avoid using a plastic Petri dish or multiwell plate, as crystals tend to form). Keep in the dark as much as possible.

5. When colour has developed to the desired extent, wash with NTMT then PBT.

6. Fix the stain with 4 % paraformaldehyde in PBS, overnight at 4 "C. If the stain is not refixed, prolonged exposure to light can cause a dark background to develop.

fixation of stain

In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

Table 3. Alternative enzyme substrates Enzyme/Substrate

Supplier

Alkaline phosphatase (AP)

I Colour

Comments

NBT/BC I P

Boehringer blue

gives the strongest signal, insoluble in water and alcohol

Fast Red

Boehringer red

strong, heat sensitive, insoluble in water, partially soluble in alcohol, will fluoresce red with a rhodamine filter set

Vector Red

Vector Labs red

weak colour reaction, heat resistant, alcohol soluble, will fluoresce red with a rhodamine filter set

Enzyme Labelled Fluorescence ( ELFTM)

Molecular Probes

green fluorescence

gives a speckled signal

Horseradish peroxidase (POD) Diaminobenzidine (DAB)

Sigma blue/black by adding Co/

The staining reactions can take from 10 min to several hours. It is therefore convenient to perform all the antibody washes the day before staining and leave the embryos in PBT overnight. This allows a full working day to monitor the development of the stain.

5.4.7 Performing Experiments with Many Different Batches of Embryos or a large Number of Probes

screening applications

T. JOWETT et al.

If you have an experiment involving more than a dozen tubes, then making changes in solutions becomes far more time consuming and boring. This can be made simpler by using 24-wel1, flat-bottomed microtitre plates and small sieves to retain the embryos. A suitable sieve can be made by cutting off the lid and the tapered end of 1.5 ml microfuge tube with a scalpel. A piece of fine nylon mesh (Nitex) can then be welded onto the top rim using a hot plate covered with aluminium foil. Such sieves are ideal for zebrafish but larger ones may be required for other embryos. They can be sterilised by soaking in bleach and washing with copious amounts of sterile deionised water. Given plenty of bench space, assemble a row of 24-well each containing different wash solutions. Embryos are retained in the sieves and moved along the assembly line through the required solutions.

This works well in initial screening of large numbers of probes or many batches of potential mutant embryos but is not recommended for those definitive hybridizationsfor publication as the washes are not as thorough and the embryos are more likely to be damaged since they are lifted clear of each solution at each change.

5.4.8 Embedding and Sectioning Whole Mount Embryos It can be useful to section the whole mount embryos after in situ hybridization. For this purpose you should choose embryos with strong signals which may even look overstained as whole embryos. Also, a little background staining may not be a problem. After fixation, the stain is reasonably stable in organic solvents, so can be embedded in wax as follows (protocol supplied by J. Slack). This protocol is designed for Xenopus embryos. For mouse embryos, one can use Histo-ClearTMrather than tetrahydronaphthalene. Note that tetrahydronaphthalene is toxic. Inhalation of the fumes and contact with skin should be avoided so all procedures should be performed in the fume cupboard.

A

hazard

1. If the embryos are in 70% glycerol, wash several times with PBT. 2. Transfer to screw cap glass scintillation vials (tetrahydronaphthalene dissolves many plastics).

3. Wash with methanol, 5 min, with isopropanol, 10 min, and then with tetrahydronaphthalene, 15 min. Replace with fresh tetrahy-

post-'SH

dronaphthalene. This clears the embryos, and you can take photographs, but take appropriate precautions against fumes, and do not spill the tetrahydronaphthalene. (The embryos can then be transferred to and stored in mineral oil indefinitely. However, you cannot embed directly from mineral oil).

4. Transfer through 1 : 1 tetrahydronaphtha1ene:paraffin wax, at 60 "C for 20 min, then paraffin wax, 3x 20 min, also at 60 "C. 5. Transfer to an embryo dish at 60 "C, place at room temperature, orientate under a dissection microscope and let the wax set.

6. Cut sections (see Section 5.6.3).

7. Dewax with Histo-ClearTM. 8. Mount under DPX mounting agent.

5.5 Double-labellingWhole Mount In Situ Hybridization This method is essentially the same as above but involves the use of differentially labelled probes and different methods of visualisation. Treatment of the embryos up to the prehybridization stage is identical. In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

optimization of signal intensity

Then a mixture of two probes is added: one labelled with digoxigenin and the other with fluorescein. It makes little difference which hapten is used for each probe as both fluorescein-12-UTPand digoxigenin-llUTP label equally efficiently and the antibodies used for subsequent visualisation work equally well. Prior to trying a double label experiment it is advisable to check each probe separately paying attention to the length of time required to obtain the final signal. Ideally, you should adjust the quantities of probe so that they will give equivalent signals in similar times. The most sensitive visualisation reaction is with alkaline phosphatase and NBT/BCIP and so this should normally be used for the probe which gives the weaker signal. The Fast Red reaction of alkaline phosphatase and the diaminobenzidine reaction for horseradish peroxidase are considerably less sensitive.

5.5.1 Hybridization 1. Prehybridize embryos at the temperature and in hybridization buffer of your choice. (See Table 1). hybridization

2. Replace prehybridization buffer with hybridization buffer containing both probes. We use 1/100 of a standard digoxigenin or fluorescein riboprobe reaction (Section 5.3.2). in 200 pl hybridization solution as a starting point. Total probe concentration should not exceed 1 pg/ml.

3. Incubate overnight at 60-70 "C in a 1.5 ml microfuge tube in a heating block or hybridization oven.

4. Follow the post-hybridization wash protocol that is appropriate for the hybridization conditions you used. (Section 5.4.3)

5.5.2 Preabsorption of the Antibodies

reduction of background signal

The antibody must be preabsorbed against the appropriate acetone powders as before. For zebrafish, add 1 yl (0.75 units) antibody to 400 yI of PBT containing 5 % sheep serum, 2 mg/ml BSA, 1 % DMSO and 1 % w/v zebrafish powder. Shake gently for at least 1 hour at room temperature then spin in a microfuge before diluting to the desired concentration. The diluted antibody is stable under these conditions at 4 "C and we have reused it up to three times.

5.5.3 Alkaline Phosphatase/horseradish Peroxidase Staining (BlueiBrown) The following is the procedure we use for zebrafish whole mounts. For larger embryos it is better to use the maleic acid buffer-based visualisation step (MABT). T. JOWETT et al.

1.

Wash off probes as usual.

2. Block with blocking solution (2 mg/ml BSA, 5 % sheep serum, 1 % DMSO in PBT) for a minimum of 60 min.

visualization AP/POD stain

3. Add 1/1000 dilution (0.75 units/ml) of preabsorbed sheep anti-

4.

digoxigenin-horseradish peroxidase Fab fragments in blocking solution. Incubate for 2 h. Wash for 2 h in blocking solution minus sheep serum (8x 15 min).

5. Incubate 2 min in 0.5 mg/ml diaminobenzidine in PBT.

6. Add 1/1000 volume of 3 % hydrogen peroxide to each incubation separately. Monitor staining and stop by rinsing thoroughly with PBT. Leave in PBT at 4 "C overnight.

7. Block with blocking solution for a minimum of 60 min. 8. Incubate for 2 h in a 1/2000 dilution (0.375 units/ml) of preabsorbed sheep anti-fluorescein-alkaline phosphatase coniugated Fab fragments.

9. Wash for 2 h in blocking buffer minus sheep serum (8x 15 min). 10. Equilibrate 3x 5 min in freshly made NTMT buffer. Stain with NBT/BCIP (4.5 pl of 75 mg/ml NBT in 70% dimethylformamide and 3.5 pI of 50 mg/ml BClP in dimethylformamide added to 1 ml of NTMT buffer).

11. Stop the reaction with washes in PBT.

12. Fix the stain in 4 % paraformaldehyde in PBS overnight. Note that diaminobenzidine (DAB) is a potent carcinogen and so should be handled with care. All disposable materials should be incinerated and contaminated glassware soaked in a strong bleach solution (6%) before rinsing thoroughly with water. One can either add the two antibodies sequentially as above or together. Also, the order of staining is not important. If the DAB reaction is performed first the hydrogen peroxide may partially inhibit alkaline phosphatase but with a good probe this is not normally a problem. However, it is important that the anti-digoxigeninhorseradish peroxidase antibody is used at a higher concentration (1/ 1000 dilution) than the alkaline phospkatase conjugated antibody. Note that the horseradish peroxidase-coniugated antibody is supplied lyophilised. Redissolve the powder in water at 150 units/ml (this is not the same titre as the AP-conjugated Fab fragments). Use at 1 in 200 dilution. Examples of zebrafish whole mount in situ hybridizations stained by this method are shown in figure 2A-D.

safety hazard

dilution of the antibodies

In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

Fig. 2 A-E. Double label in situ hybridization (A and C) Zebrafish embryo whole mounts probed with digoxigenin-labelled antisense RNA fo pax-b (Krauss et a/., 1991) and fluoresceinlabelled antisense RNA for krx-20 (Oxtoby and Jowett, 1993).The pax-b signal is visualised with antidigoxigenin horseradish peroxidase and diaminobenzidine (brown).The krx-20 signal is visualised with antifluorescein alkaline phosphatase and stained with NBTBClP (blue). (B and D) Zebrafish embryo whole mounts probed with digoxigeninlabelled antisense RNA for krx-20 and fluoresceinlabelled antisense RNA for pax-b. The krx-20 signal is visualised with antidigoxigenin horseradish peroxidase and diaminobenzidine (brown).The pax-b signal is visualised with antifluorescein alkaline phosphatase and stained with NBTBClP (blue). (E) A zebrafish whole mount embryo probed with digoxigenin-labelled pax-b antisense RNA and fluorescein-labelled krx-20 antisense RNA. The signals are visualised sequentially with anti-digoxigenin alkaline phosphatase/NBT-BCIP (blue) and anti-fluorescein alkaline phosphatase/Vector RedTM(red). In C, D and E the embryos are equilibrated with 100% glycerol, dissected away from the yolk and flattened on a microscope slide underneath a coverslip

Fig. 3 A-C. Overlapping and nonoverlapping signals in double label in situ hybridization (A and B) Zebrafish embryo whole mounts probed with fluorescein-labelled krx-20 antisense RNA and digoxigenin-labelled antisense RNA for hox-62 (Yan, Jowett and Postlethwait unpublished) respectively. The km-20 signal is visualised with antifluorescein alkaline phosphatase/Fast Red and the OX-62 signal with anti-digoxigenin alkaline phosphatase/NBTBClP (C) A zebrafish embryo probed with a mixture of the probes in A and B and the visualised sequentially with NBT-BCIP and Fast Red. krx-20 is present in two stripes (rhombomeres 3 and 5). hox-62 is expressed in three stripes (rhombomeres 3,4 and 5) in decreasing amounts. The combination of the two probes produces different coloured signals in the three rhombomeres

T. JOWETT et al.

b

Fig. 3 A-C

In Situ Hybridizationto Embryo Whole Mounts and Tissue Sections

5.5.4 Sequential Alkaline Phosphatase Staining (Blue/Red)

4 visualization sequential AP

1. Wash off probes as usual. 2. Block with blocking solution (2 mg/ml BSA, 5 % sheep serum, 1 % DMSO in PBT) for 60 min. 3. Add 1/2000 dilution of meabsorbed sheeD anti-fluoresceinalkaline phosphatase co'njugated Fab fragments in blocking solution. Incubate for 2 h.

4. Wash for 2 h in blocking solution minus sheep serum (8x 15 min).

5. Equilibrate 3x 5 min in NTMT buffer. Stain with NBT/BCIP 6. Stop reaction with washes in PBT and fix in 4 % paraformaldehyde in PBS. 7. Rinse in PBT and heat to 65 "C for 30 min to inactivate the alkaline phosphatase. An alternative to heat inactivation is to incubate in 100 mM glycine pH 2.2; 0.1 % Tween-20 for at least 30 min. followed by washing thoroughly in PBT.

8. Block with blocking solution for 60 min. 9. Incubate for 2 h with a 1/2000 dilution of preabsorbed sheep anti-digoxigenin-alkaline phosphatase Fab fragments.

10. Wash 8x 15 min in PBT. 11. Equilibrate 3x 5 min in 100 mM Tris-HCI pH 8.2. 12. Stain with Fast Red (Boehringer). Dissolve one tablet in 2 ml of 100 mM Tris-HCI pH 8.2. The staining solution should be made fresh and used within 30 min. There are a few undissolved particles which may be removed by filtering. Incubate embryos in staining solution at room temperature until colour develops. The yolk takes up the staining solution becoming quite yellow. This is reduced, but not eliminated in the subsequent washes.

13. Stop the reaction by rinsing in PBT and fixing in 4 % paraformaldehyde/PBS.

choice of stain

T. JOWETTetul.

We originally used the Vector Red alkaline phosphatase staining kit (Jowett and Lettice, 1994) but this gives a weaker signal than Fast Red (Hauptmann and Gerster, 1994).The Vector Red precipitate is stable to heat whereas the Fast Red product rapidly fades if heated. An equivalent product is available from Sigma (Sigma Fast-Fast Red/TR/ naphthol AS-MX tablet sets F4648). This is a cheaper alternative but we have not evaluated it on vertebrate embryos and do not know the stability of the red precipitate to heat. An example of a zebrafish whole mount double label in situ hybridization stained with NBT/BCIP and Vector Red is shown in figure 2E. It is best to stain with NBT/BCIP first. If the first alkaline phosphatase is incompletely inactivated, when the NBT/BCIP is added a blue colour will develop on top of the Fast Red and mask it.

Fig. 4 A-D.Double label in situ hybridization to embryo sections. (A) Zebrafish embryo section probed with digoxigenin-labelled antisense RNA for CoMaI (the gene for alpha (1) collagen type (11) which is expressed in notochord, referenceYan eta/., 1995)and fluorescein-labelled antisense RNA for a gene as yet uncharacterised but expressed in the somites (Van unpublished).The Col2a 1 signal is visualised with ant-digoxigenin horseradish peroxidase/DAB (brawn) and the somite signal with anti-fluorescein alkaline phosphatase/NBT-BCIP (blue). (B and D) Zebrafish embryo section probed with the same probes as in (A) but visualised by sequential incubation and staining with anti-digoxigenin alkaline phosphatase/ Fast Red (Boehringer)and anti-fluorescein alkaline phosphatase/NBT-BCIP (blue). (6) is viewed with the light microscope and (D) is viewed by epifluorescence with a rhodamine filter set. The notochord is red in (6) and fluoresces strongly in (D).Note the background fluorescence in (D)and how it is quenched by the blue/black precipitate in the somites. (C) Zebrafish embryo sagittal section probed with digoxigenin-labelled antisense RNA for Col2a I and the signal visualised with antidigoxigenin alkaline phosphatase/ELFTM. The section is viewed by epifluorescence with a DAPl filter set. There is green/yellow fluorescent precipitate in the notochord

In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

5.5.5 Special Considerations

overlapping signals

T. JOWETT et ol.

Double label in situ hybridizations work best for non-overlapping signals. The blue/brown and blue/red colour reactions provide good contrast between signals. However, where target transcripts are colocalised it is more difficult to get the balance right between the two signals. For instance, the NBT/BCIP blue stain can be very intense and easily hide the red or brown colour. However, if the staining reactions are carefully monitored and the weaker red staining performed first, then the blue reaction can be stopped before it completely masks the red, while there are three different colours: red, blue/red and blue. In this case it is essential that special care is taken to inactivate the first alkaline phosphatase prior to incubation in the second antibody. Figure 3 shows a double label in situ hybridization with overlapping probes identified with Fast Red and NBT/BCIP. Fast Red and Vector Red both fluoresce strongly with a rhodamine filter set so if the tissue is only weakly staining examination by epifluorescence can enhance the signal. We have tried to perform whole mount double label in situ hybridization using fluorochromes but with limited success. Sheep antidigoxigenin conjugated with fluorescein or rhodamine are available (Boehringer) but these alone are not sensitive enough to identify digoxigenin-labelled probes. Further amplification steps are required. Molecular Probes (Eugene) supply an alkaline phosphatase substrate which initially is non-fluorescent but when activated by the enzyme will fluoresce yellow/green. This so-called Enzyme Labelled Fluorescence (ELFTM) can be used as an alternative to the standard substrates and will provide a signal without further amplification. However, the fluorescent compound is deposited in large crystals which give a speckled appearance to the signal. The embryos are extensively washed in a 30 mM Tris-HCI, 150 mM NaCl pH 7.5 prior to staining in diluted ELFTM substrate. The staining reaction takes 30 min to 2 hours. It is stopped with 25 mM EDTA, 0.05 % Triton X-100 in PBS at pH Z2. With zebrafish embryos it is not necessary to add 1.0 mM levamisole. The large crystals that form may be reduced by replacing Tween20 in the antibody incubations and washes with Triton X-100. The embryos must be mounted in a special mounting medium, provided in the kit. This mountant preserves the ELFTMsignal better than the usual glycerol-based aqueous mountants for fluorochromes. O n zebrafish whole mounts it is not normally necessary to increase the titre of the alkaline phosphatase-conjugated antibody when using the ELFTMsubstrate. Note also that the yellow/green fluorescent signal is easily quenched by the dark blue precipitate of NBT/BCIP in double labelling experiments with overlapping signals. An example of the use of the ELFTMsubstrate on a zebrafish embryo section is shown in figure 4C.

5.6 In Situ Hybridization to Tissue Sections Although, it is often easier and quicker to perform whole mount in situ hybridizations it is ultimately necessary to examine sections to obtain single cell resolution. These can be made from stained embryos (Section 5.4.8) or from fixed tissue followed by in situ hybridization. The procedure for performing in situ hybridization to sections is outlined below. The principles are the same as for whole mount embryos but there are some special considerations. The tissue or embryo must be fixed and embedded in a suitable medium for cutting sections. Tissue can be frozen and sections cut with a cryostat. This is quicker but, in general, produces poorer sections since for small embryos the sections must be relatively thick. The preferred embedding medium is paraffin since embryos can be fixed and stored in ethanol or methanol prior to embedding and once embedded the blocks can be kept indefinitely at room temperature or at 4 "C. Most tissues or embryos can be embedded in the same manner as long as times of incubations are adjusted according to the size of the material. Paraffin sections are cut with a standard microtome preferably with a disposable blade holder. They are transferred to microscope slides as single sections or as ribbons representing the entire embryo or tissue. Thus adjacent sections can be probed with different probes or sections of entire embryos can be probed simultaneously in a single hybridization. Embryos can be arranged while embedding to allow sectioning in a particular orientation. If the embryos are small like zebrafish, then many embryos can be embedded in a single block so that most orientations are represented in each section. It is important to treat the slides which are to receive the sections so that they do not wash off during the hybridization and visualisation steps. Addition of probe and antibody is directly to the sections on the slide underneath a coverslip and so care must be taken to prevent the slides from drying until the end of the procedure. The original method involved the use of radioactive probes and visualisation by dipping in photographic emulsion. However, most laboratories now use nonradioactive methods since they are safer, more convenient, offer better resolution, are less expensive and are quicker.

5.6.1 Preparing TESPA-Treated Slides Slides may be treated with gelatine, poly-lysine or 3-aminopropyltriethoxysilane (TESPA) to ensure that the sections stick to the slides throughout the hybridization and subsequent washes. TESPA is the treatment we prefer since it is the easiest to perform. In contrast, there is no necessity to treat coverslips which are used straight from the box. Plain glass slides are the best to use as those with frosted ends may draw out the hybridization solution from under the cover slip. The penalty is that plain slides must be marked with a diamond pencil whereas frosted glass can be marked with a normal pencil. In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

@ slide preparation

1.

Load slides into a glass slide holder.

2. Rinse in 70% ethanol. 3. Rinse in 1N HCI.

4. Rinse in distilled water. 5. Rinse in 95% ethanol.

6. Allow to dry in oven at 150 "C. 7, In a fume hood, dip slides for 10 sec. in 2% 3-aminopropyltriethoxysilane (TESPA) in acetone at room temperature. 8. Dip slides twice in acetone for 10 sec. 9. Dip slides in distilled water for 10 sec. 10. Dry at 42 "C.

11. They can be stored in their original boxes at room temperature.

5.6.2 Fixation and Embedding Embryos or Tissue Any of the fixatives described for whole mount in situ hybridizations can be used (Section 5.4.1). The same considerations about time of fixation and size of embryo or tissue apply. The protocol below is what we use for zebrafish embryos.

1. fixation & embedding

Fix embryos in paraformaldehyde ( 4 % in PBS), 12h to overnight, at 4 "C.

2. Rinse embryos in PBS. 3. Dechorionate with watchmakers forceps. 4.

Dehydrate with several changes of 100% methanol and store at -20 "C.

5. Equilibrate with 100% ethanol. 6. Incubate 5 min twice with Histo-ClearTM.Xylene or toluene can be used but we use Histo-ClearTMas it is considerably less hazardous. It has also been suggested that xylene-treated tissue is unsuitable for in situ hybridization to sections using digoxigenin-labelled probes (Tautz et al., 1992).

7. Incubate 30 min three times with melted paraffin at 60 "C. For zebrafish embryos this is most conveniently performed in the glass vials used for storage of the embryos -20 "C. We use a dri-block with aluminium inserts with holes to take the glass vials. This can be placed in a fume cupboard to remove the toluene vapour. T. JOWEll et al.

8. The exact manner of embedding the tissue or embryos depends on the nature of the microtome and block holder one has available. Most microtomes will accommodate plastic embedding cassettes which fit into stainless steel or disposable plastic moulds.

9. Preheat metal moulds and plastic cassettes on an aluminium tray at 60 "C in an oven or on a warming plate.

10. Keep wax in glass vials in the heating block. 11. Using a glass Pasteur pipette warmed in a Bunsen flame transfer the embryos to the metal mould.

12. Fill the well with wax and swirl the embryos to the centre. 13. Top up with more wax and add the plastic holder. The wax should cover the holes in the holder. A pencil marked paper label can be stuck in the wax.

14. Remove from the warming plate to a cool surface and allow to set.

15. Cool on ice and then release the cassette holder with a scalpel. 16. Store in plastic bags or in the cardboard embedding cassette holders. Single mouse embryos can be embedded in a similar way but orientated before the paraffin solidifies. This can be done by arranging the warming tray under a dissecting scope and slowly allowing the wax to cool by placing the mould on a cool surface. Support the embryo in the correct orientation with a fine syringe needle until the wax begins to set and then remove the needle. The embryo should retain its orientation.

orientate embryos

5.6.3 Cutting Paraffin Sections Cutting sections is a skill acquired through practice. If there is anyone within your institution who has experience in cutting sections it is well worth asking them for help. In general, bigger pieces of tissue are easier to section than small ones. A problem with zebrafish embryos is that the yolk tends to fall away and does not adhere to the slide.

1.

Remove the metal or plastic mould from the block. This is most easily done by chilling the block.

2. Cut off excess wax and trim to a trapezoid shape with the narrow part towards the top. The block should be symmetrical otherwise the section ribbons will be curved.

cutting sections

3. Cool the wax blocks on ice. If the block is too warm alternate sections will be thick and thin. If too cold the sections will not form a ribbon. In Situ Hybridizationto Embryo Whole Mounts and Tissue Sections

4.

Lock the rotary wheel of the microtome so that the cassette holder does not move while you mount the blade. Take a new disposable blade and rinse in Histo-ClearTMto remove the protective oil. Keep a glass Coplin jar of Histo-ClearTMnext to the microtome for this purpose. Secure the disposable blade in the blade holder and mount in the microtome.

5. Mount the block and adjust the forward advance to give enough travel to cut the whole block.

6. Align block using the adjustments so that the surface is parallel with the blade.

7. Cut sections at 5-8 microns using a paint brush or pair of forceps to hold the ribbon. Place labelled slides (pencil or diamond writer) on warming plate at about 40 "-45 "C. If there is scoring or splitting of the sections at right angles to the blade, the blade edge is damaged or there are hard particles in the wax or tissue. If the sections roll into a tight coil instead of remaining flat, the blade is blunt or the sections are too thick.

8. Cover the slide with sterile water and layer the sections slide neatly on the surface.

9. Remove excess water with a Pasteur pipette and allow to dry down onto the slide. The wax stretches and wrinkles are removed. Avoid having the slides too hot.

10. Leave to dry completely at 37 "C overnight.

use a floating out bath

Alternate sections can be placed on separate slides in which case the ribbons may be first laid on a clean sheet of aluminium foil and a scalpel used to separate the sections. A floating out bath can be used for first removing the creases in the paraffin sections before transferring them to a slide by bringing the slide into contact with the section from below the surface of the water. However, one is always worried that the water in such a water bath will be contaminated with RNases so a warming tray is the safer option. In optimising conditions for a particular probe one may wish to try different modifications of the procedure and therefore a single section per slide may be hybridised. If so, it is a good idea to put the section on the end of the slide furthest away from the frosted or labelled end. This allows the slide to be dipped in the minimum volume of wash solution in polypropylene Coplin jars or slide mailers.

5.6.4 Pretreatment and Hybridization of Sections The sections must first be dewaxed in an organic solvent such as toluene, xylene or Histo-ClearTM.They can then be refixed but this is optional. Some embryos which have pigment like Xenopus must be bleached but otherwise omit this step. The tissue is permeabilised with proteinase K as with whole mounts. A prehybridization step is usually T. JOWETT eta/.

not necessary, but if included, all the prehybridization solution should be removed with filter paper before adding the probe solution to avoid dilution of the probe.

1.

Load slides into a glass rack and rinse twice in Histo-ClearTM for 5 min.

2. Rehydrate through ethanol series as follows: 2x 30 sec. in 100% ethanol; 1 x 30 sec. in 95 % ethanol; 1x 30 sec. in 90 % ethanol; 1 x 30sec. in 70 YOethanol; 1x 30 sec. in 50 % ethanol. 3. Air dry. Store in a slotted, microscope slide box at -20 or 4 "C. 4. Wash with PBT twice for 5 min each.

W

hybridization of sections

5. Fix with 4 % paraformaldehyde in PBS for 20 min. This step is optional.

6. Wash with PBS, twice for 5 min. 7. Bleach with 6% hydrogen peroxide in PBT, 1 h. This is only necessary if pigmentation is a problem, as with Xenopus embryos. Bleaching can be performed after ISH and visualisation rather than before (Harland, 1991).

8. Wash three times with PBT, 5 min each. 9. Treat with 5-10 pg/ml proteinase K in PBT, for 15 min. The time

bleaching to remove pigment

must be determined empirically for the particular type of tissue and batch of enzyme.

10. Wash with PBT, twice for 1 min. 11. Fix with 4 % paraformaldehyde in PBS for 20 min 12. Wash with PBT, twice for 5 min. 13. Quickly pass through a methanol series from 30% to loo%, then air-dry. This step is optional but does ensure that if a prehybridization step is omitted the probe efficiently infiltrates the section.

-

2.5 p k m 2 hybridization mix with probe to slides and lower a coverslip on top so that the liquid spreads over the sections. Take particular care to eliminate air bubbles. Add the hybridization solution along one side of the sections. Place the edge of the coverslip in the solution and gently lower over the remaining sections using forceps or a scalpel blade to support the coverslip. The hybridization solution should wet the sections eliminating air as the coverslip is lowered.

14. Apply

15. Place slides horizontally in a square plastic Petri dish containing tissue soaked in a solution of formamide and SSC with the same concentration as the hybridization solution. Prewarm the box for the slides and also the probe solution to the hybridization temperature. Enclose the box in a plastic bag and incubate overnight at 60 "-70 "C depending on hybridization solution used. In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

5.6.5 Washing of Sections and lmmunodetection of Probe The washing procedure depends on the hybridization conditions. We use the following protocol for sections treated with zebrafish hybridization buffer. The antibody concentrations used on sections are generally higher than those used for whole mounts but also depend on the native of the enzyme conjugant (Table 4). 1.

washing

8t visualization

Transfer the slides to a Coplin iar containing 50% formamide and 2x SSC prewarmed to 65 "C for 15 min. There should be sufficient solution to immerse the coverslip. The coverslip should fall away.

2. Wash for a further 30 min in 50% formamide 2x SSC at 65 "C. We perform this and subsequent washes on the shaker in the Dual Hybridization oven with gentle agitation.

3. Wash twice in 25% formamide, l x SSC, 0 . 5 ~PBS at 65 "C for 30 min. This is the stringent wash. 4. Wash in PBS at room temperature for 5 min. 5. Transfer the slides to a square Petri dish as used for the hybridization containing tissue soaked in l x PBS. Add enough blocking solution ( l x PBS containing 0.1 % Tween-20, 5 % sheep serum and 0.2% BSA) to cover the sections and incubate at room temperature for 60 min. Table 4. Double labelling methods for in situ hybridization to sections Sequential AP staining Probes

Sequental AP and POD staining

Sequental AP and POD staining

20,5% sheep

20,5 % sheep

U/ml (1 :15)

U/ml (1500)

Mix of fluorescein and digoxigenin labelled antisense RNAs

Hybridization

zHybe 60-70 "C

Block solution

PBS, 0.1 '30Tween20,5 % sheep serum, 0.2% BSA

First antibody incubation Stain

sheep antidigoxigenin-AP 0.5 U/ml (1: 1500)

I NBT/BCIP

I DAB

Inactivation

30 min in 100 mM glycine pH 2.2 at room temperature

not necessary

Second antibody incubation

sheep antidigoxigenin-AP 0.5 U/ml (1:1500)

sheep antifluorescein-AP0.5 U/ml (1:1500)

I Fast Red not necessary I

Stain T. JOWETT et al

I Fast Red

I NBT/BCIP

sheep antidigoxigenin-POD 10 U/ml (1:15)

I DAP

6. Remove the block solution with filter paper and add the diluted antibody, preabsorbed with the appropriate acetone powder. Use 10-50 PI depending on size of coverslip required to cover the sections. Incubate 30-60 min at room temperature or overnight at 4 "C in a humid chamber containing tissue soaked with PBS. If left overnight use a coverslip.

7. Wash four times for 15 min in a Coplin jar with l x PBT at room temperature.

8. Wash at room temperature for 15 min in the appropriate buffer for the staining reaction. 0

0

For visualising alkaline phosphatase with NBT/BCIP use NTMT. Change the solution at least three times before adding stain to ensure the pH is high. 2 mM levamisole can be included in the NTMT to inhibit endogenous phosphatases, but we have not found this to be needed in zebrafish, mouse and chick. Levamisole should be included in experiments on Xenopus sections. For visualising alkaline phosphatase with Fast Red use 100 mM Tris-HCI pH 8.2. For visualising horseradish peroxidase with DAB use PBT.

9. The staining reaction should be performed in the dark either by dipping the slide in a minimum volume of staining solution or by putting a drop on the sections and coverslipping.

10. Stain until the desired signal strength is achieved (usually 45-60 min). Stop the reaction by washing with PBT.

11. For permanent mounts it is best to fix the stain by incubating in 4 % paraformaldehyde in PBS for 10-20 min at room temperature.

12. If the staining reaction is DAB or NBT/BCIP use a non-aqueous mountant. Wash the slides in deionised water and dehydrate through an ethanol series 50%, 70%, 96%, 100% 10 min in each and then twice in Histo-ClearTMTM for 10 min. Add a drop of DPX mountant and a coverslip.

13. If Fast Red is the stain used then we recommend that you use an aqueous mountant such as UVinertTM aqueous. This will also allow the use of epifluorescence to visualise the signal. See figure 4D. Examples of double labelling on zebrafish sections with NBT/DAB and NBT/Fast Red are shown in figure 4A and B respectively.

In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

5.7 Troubleshooting

\

troubleshooting

We would hope that all the methods described here will give you at least partial success in most tissues or organisms. However, here are some tips as to experiments and controls to try if your results are not satisfactory.

No Signal

\

This could result in several ways. The tissue may not carry the target RNA or the RNA has been degraded by RNases or lost from the tissue. The probe may be degraded. The antibody may be nonfunctional. The substrates for the staining may be non-functional. a. Are the sections or tissues of the correct developmental stage? b. Is the riboprobe intact? Check integrity probe by running on an agarose minigel. c. Use.anotherprobe as a positive control if you have one available. d. Increase the probe concentration and lower the temperature of hybridization. e. Check the antibodies by applying a small amount ( 1 ~ 1 )of riboprobe to a nitrocellulose or nylon filter, UV-crosslinking to bind the RNA and following the procedure for antibody visualization. f. Try increasing the titre of antibody twofold or increasing the length of incubation. g. Check the staining reagents by applying a small amount (1pI) of diluted antibody to a nitrocellulose or nylon filter and following the staining procedure. High Background This could result in two ways. The probe could be hybridizing nonspecifically or the antibody could be binding non-specifically. a. To determine whether it is a probe or antibody problem, perform a control hybridization with no probe but normal antibody visualization. If there is no signal the background results from the probe. If the background signal persists it is because the antibody titre is too high or is inadequately preabsorbed or the non-specific binding sites in the tissue is incompletely blocked. b. If the problem lies with the probe. Try the following: Prehybridize for longer. Lower the concentration of the probe. Increase the stringency by raising the temperature of hybridization and/or the for mamide concentration in hybridization mix. c. If the problem lies with the antibody. Try the following: Increase the blocking time. Use a lower antibody titre and increase the staining period.

T. JOWElT et 01.

Poor Morphology

This could arise for a variety of reasons: a. Insufficient fixation: increase the fixation time or change to a stronger fixative. b. Excessive proteinase K treatment: reduce incubation time or concentration of enzyme and increase post-proteinuse K fixation. c. Reduce temperature of hybridization and try different pH for the buffer.

Acknowledgements We should like to thank David Wilkinson, Philip and Anita Ingham, Stefan SchulteMerker, Jonathan Slack, Laura Lettice, Henrique Domingos, David Tannahill, Peter Holland for sharing their experiences of performing in situ hybridizations.TJ, MM, AA were supported by grants from EMBO, the Wellcome Trust and the North of England Cancer Research Campaign. YY was supported by grant number NIH 1 PO1HD22486 and NIH 1R 0 1 RR/RG10715.

References Forbes, Z. and Ingham, P W. (1993) Drosophila embryos. In ,,Essential Developmental Biology: A Practical Approach" pp 1-13. Ed. C.D. Stern and PW. Holland. Publ. IRL Press, Oxford. Harland, R. M. (1991) In situ hybridization: an improved whole mount method for Xenopus embryos. In Methods in Cell Biology 36: 685-695. Hauptmann, G. and Gerster, T. (1994)Trends in Genetics 10: 266. Two-color wholemount in situ hybridization to vertebrate and Drosophila embryos. Henrique, D., Adam, J., Myat, A., Chitnis, A,, Lewis, J. and Ish-Horowicz, D. (1995) Nature 375: 787-790. Expression of a Delta homologue in prospective neurons in the chick. Jowett, T. and Lettice, L. (1994)Trends in Genetics 10: 73-74. Whole-mount in situ hybridization on zebrafish embryos using a mixture of digoxigenin and fluorescein-labelled probes. Krauss, S., Johansen, T., Korzh, V. and Fjose, A. (1991) Development 113: 1193-1206. Expression of the zebrafish paired box gene pax during early neurogenesis. Lamb,T. M., Knecht, A. K., Smith, W. C., Stachel, S. E., Economides, A. N., Stahl, N., Yancopolous, G. D. and Harland, R. M. (1993) Science 262: 713-718. Neural induction by the secreted polypeptide Noggin. Oxtoby, E. and Jowett, T. (1993) Nucl. Acids Res. 21: 1087-1095. Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Strahle, U., Blader, P, Adam, J. and Ingham, P W. (1994) Trends in Genetics 10: 75-76. A simple and efficient procedure for non-isotopic in situ hybridization to sectioned material. Tautz, D., Hulskamp, M. and Sommer R.J. (1992)Whole mount in situ hybridization in Drosophila. In "In situ hybridization: A Practical Approach" pp 61-73. Ed. D.G. Wilkinson Publ. IRL Press, Oxford. Wilkinson, D.G. (1992)The Theory and Practice of In situ Hybridization. In "In situ hybridization: A Practical Approach" pp 1-13. Ed. D.G. Wilkinson Publ. IRL Press, Oxford. Yan, Y.-L., Hatta, K., Riggleman, B. and Postlethwait, J. H. (1995) Developmental Dynamics 203: 363-376. Expression of a type I1 collagen gene in the zebrafish embryonic axis. In Situ Hybridization to Embryo Whole Mounts and Tissue Sections

In Situ Hybridization Melody Clark Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

In Situ Hybridization at the Electron Microscopic Level F. WACHTLER, C. SCHOFER and K. WEIPOLTSHAMMER

6.1 Int rodu ct ion Like no other approach, in situ hybridization at the electron microscopic (EM) level offers the fascinating possibility of correlating the distribution of specific nucleic acids to the ultrastructure of cells. This has been made possible by the development of nonautoradiographic labelling techniques for nucleic acids and also the availability of techniques for the detection of the labelled hybrids. These non-autoradiographic methods produce a much more precise spatial resolution when compared with radioactive protocols. Another important factor in the successful implementation of in situ hybridization techniques at the EM level was the development of hydrophilic acrylic resins, such as Lowicryl or LR White. However, in situ hybridization at the EM level also has limitations and shortcomings:

1. The staining contrast obtained with in situ hybridization protocols is

principal problems

lower than in conventional electron microscopy, making the interpretation of structures sometimes difficult. 2. Only a very small fraction of the nucleic acid to be detected is present at the surface of an EM section. Therefore the observed signal in a single section is generally not representative of the overall distribution of the nucleic acid sequence under scrutiny. 3. Finally, the signa1:background ratio may be rather low. We believe that these limitations notwithstanding in situ hybridization at the EM level can produce meaningful results if:

1. The limitations are kept in mind 2. Appropriate controls are carried out (we find it particularly helpful to perform light and electron microscopic experiments in parallel)

3. Statements are not based on single micrographs but on a statistical analysis of a large number of individual images. In this chapter, we shall outline the procedures and protocols for in situ hybridization at the EM level as used in our laboratory. One proM. Clark (Ed.),In Situ Hybridization.@ Chapman & Hall, Weinheim. ISBN 3-8261-0038-7

tocol for each procedure will be described although a variety of other methods exist. We feel that it is more helpful for the beginner to start with one protocol that has been found to work reliably instead of having to select from a wide variety of different protocols, the advantages and disadvantages of which are difficult to judge. In addition, we will address the limiting points mentioned above in some detail. We have been working mainly with ribosomal DNA (rDNA, ribosomal gene repeat, ribosomal RNA genes) and ribosomal RNA (rRNA) and shall refer mainly to these types of nucleic acids. Furthermore, despite the use of very similar protocols, the results obtained by different groups working in this field are remarkably different and controversial and may serve as an instructive example for the difficulties which arise when interpreting the outcome of in situ hybridizations (Thiry and Thiry- Blaise, 1989, 1991;Wachtler eta/., 1992;Wachtler and Stahl, 1993)

6.2 Materials and Chemicals 6.2.1 Buffers and Reagents BSA: 1 % BSA in 1xPBS; adjust one part to pH 7.4 another to pH 8.2. Use a molecular biology grade of BSA. EGTA buffer: 1mM EGTA in 1xPBS

+ 100ng/pl salmon sperm DNA 100ng/pl yeast tRNA + 10 % dextran sulfate in 50 % formamide in 2xSSC

Hybridization mixture: 2ng/pl labelled probe

+

Paraformaldehyde: 4 % in 1xPBS (should be prepared immediately before use, for a protocol see 6.4.1)

PBS stock solution 10xPBS: 1.5M NaCl + 167mM Na2HP04+ 17mM NaH2P04. Adjust to pH 7.2-7.4 after diluting to 1 x strength. PBS/MgCI2: 50mM MgCI2in 1xPBS PBT: 0.05% Tween 20 in 1xPBS; adjust one part to pH 7.4 another to pH 8.2. Pepsin solution: 0.1 % in 0.01 N HCI. Prehybridization mixture: 200ng/pl denatured salmon sperm DNA + 200ng/pl yeast tRNA + 0.1 % Ficoll400 + 0.1 % BSA + 0.1 % PVP in 50% formamide in 2xSSC Proteinase K solution: 0.5 pg/ml in Tris/CaCI2 buffer RNase A solution: 100 pg/ml in 2xSSC RNase H buffer: lOOmM NaCl + 50mM TrisHCl + lOmM MgCI2 RNase H: 0.5U/pl in RNase H-buffer Saponin: 0.1 % saponin in EGTA buffer

SSC stock solution: 2OxSSC: 0.3M sodium citrate + 3M NaCl Stringency wash solution: 50 % formamide in 2xSSC Tris/CaCI2 buffer: 20mM Tris F. WACHTLER e t d

+ 2mM CaCI2

6.2.2 Chemicals Reagent anti- avidin/Biotin anti- biotin anti- digoxigenin antibody, rhodamine- labelled anti- digoxigenin antibody, unlabelled anti- goat/5nm gold anti- rabbit antibody, rhodamine- labelled anti- sheephrn gold anti- sheep/rhodamine avidin/FITC avidin/POX bio- dUTP bovine serum albumine dATP

dCTP dextran sulfate

dGTP diaminobenzidine DIG labelling and detection kit digoxigenin- dUTP (dig- dUTP)

dTTP EGTA Ficoll400 formamide Glutaraldehyde for electron microscopy gold grids hexanucleotide primers LR White Resin (medium grade) for electron microscopy Nick translation and labelling kit pepsin polyvinylpyrrolidone proteinase K RNase A RNase H salmon sperm- DNA saponin Taq- polymerase yeast- tRNA

Supplier Vector Vector

Cat no BA- 0300

Boehringer Boehringer Amersham Southern Biotech Chemicon Chemicon Vector Vector Boehringer Boehringer Boehringer Boehringer Sigma Boehringer Sigma Boehringer Boehringer Boehringer Sigma Sigma Loba

1207 750 1214 667 RPN 436 4010- 03

SP- 3000

GAB 342 AP 147R A- 2011 A- 2004

1093 070 775 827 103 977 104 035 D- 7140 104 094 D- 5637 1093 657 1093 088 104 272 E- 4378 F- 2637 47408

Merck Agar Aids Boehringer London resin

12179.0025 G2202A 1277 081

Boehringer Boehringer Sigma Boehringer Boehringer Boehringer Boehringer Sigma Boehringer Sigma

976 776 108 057 P- 5288 161 519 109 142 786 349 223 646 S- 2149 1146 165 R- 8759

6.2.3 Equipment A variety of general laboratory equipment is used in these protocols such as variable temperature incubators and waterbaths (alternatives for these include hybridization ovens or an adapted PCR thermocycler e.g. a flat heating block), microscope slides and coverslips, micropipettes etc all of which are available from most laboratory equipment suppliers. The EM analysis package is the Sigmascan Scientific Measurement Programme from Jandel. In Situ

Hybridization at the Electron Microscopic Level

6.3 Labelling Nucleic Acids for In Situ Hybridization We have only used DNAfor in situ hybridization at the EM level. In our hands the best labelling method is still nick-translation, resulting in probes with the lowest error level of all methods and lowest background in in situ hybridization experiments. For a full set of DNA probe labelling protocols see Appendix I. For quantification of probe see Appendix (I.

6.4 Preparing Tissue for Electron Microscopic In Situ Hybridization

choice of biological material

F. WACHTLER e t a /

The first and decisive step in performing successful in situ hybridization is the choice of the right biological material. We should like to give an example for this taken from our own area of interest, namely ribosomal genes and nucleoli. Nucleolus morphology varies greatly with cell type, but the same building materials or components are always present, namely fibrillar centers plus a dense fibrillar component and a granular component (for a review on nucleolar ultrastructure see e.g. Wachtler and Stahl (1993)).If one needed to distinguish in which of the three components a particular nucleic acid sequence is located, one should try to use cells with big nucleoli where the components form large and spatially ordered territories as opposed to those cells where nucleoli are small and/or nucleolar components are chaotically intermingled, which is the case in most types of cells (see fig 1). A good example of an ordered nucleolus is found in human Sertoli cells (see fig. 2), because it contains the nucleolar components in an invariable spatial order: Almost all cells contain only one large fibrillar center which is located at the periphery of the nucleolus. The fibrillar center is surrounded by a shell of dense fibrillar component forming a three-dimensional network of sheets and strands. This mesh of dense fibrillar components extends away from the fibrillar center, lining a block of granular component. Thus the nucleolar components are spatially separated and label can be attributed to either of these components unequivocally, which is not the case in most other cells. Furthermore, under favorable conditions, the nucleolar components can be seen in the light microscope.

Fig. 1. Nucleolus of a human, PHA- stimulated lymphocyte. The nucleolar components are highly intermingled. Bar: 100 nm Abbreviations: c...chromatin, d...dense fibrillar component, f...fibrillar center, g...granular component Fig. 2. Nucleolus of a human Sertoli cell. The nucleolar components are spatially ordered. Bar: 1 pm Abbreviations: c...chromatin, d...dense fibrillar component, f...fibrillar center, g...granular component

6.4.1 Fixation We use a mixture of glutaraldehyde and formaldehyde as a fixative. The higher the glutaraldehyde content, the better the preservation of ultrastructure, but the weaker the in situ signals. Formaldehyde will

fixation

In Situ Hybridization at the Electron Microscopic Level

generally improve contrast and signal, but may cause structural damage. The fixative solution should be prepared immediately before use, since paraformaldehyde will change into formic acid and methanol thus altering the pH of the solution. Never use OsO4 for fixation. We recommend the following fixation protocol as a starting point, although conditions may need to be optimised for different tissues:

A

use fume cupboard

1. Prepare a 0.2M phosphate buffer by dissolving 0.643g NaH2P04and 2.21 g Na2HP04in lOOml distilled water. 2. Depolymerize 5g paraformaldehyde in lOOml distilled water by adding a drop of 0.1N N a O H and heating the solution up to 70 "C. This step should be carried out in a fume hood, since formaldehyde is toxic and an irritant. As soon as the solution becomes clear, place on ice. Filter using ordinary filter paper. 3. Mix lOml of a commercially available glutaraldehyde solution (electron microscopy grade), lOml of the paraformaldehyde solution and 75ml of the phosphate buffer.

4. pH the solution to 7.3 using HCI or NaOH. 5. Adjust the final solution volume to 100ml. This will result in a solution containing 2.5 % glutaraldehyde, 0.5 % formaldehyde dissolved in 0.15M phosphate buffer. 6. Tissue or cells should be fixed in this solution at 4 "C for half an hour. Tissue should be cut into pieces not exceeding 0.5 mm in diameter.

7. After fixation the fixative should be removed by rinsing the cells or the tissue in several washes of phosphate buffer until the smell of aldehydes can no longer be detected.

8. Perform a dehydration series in 30%, 60%, 70%, 80%, 90%, 95% and 100% ethanol for half an hour each on a shaker at room temperature.

6.4.2 Embedding, Cutting and Mounting Several hydrophilic resins are available, we prefer LR White. A protocol and hints for this resin are outlined below:

1. The cells or tissue fragments are passed through the following series of solutions: embedding

F. WACHTLER et 01.

3 parts 100 % ethanol and 1 part LR White Medium Grade for 24 hours at 4 "C. Equal amounts of LR White and 100% ethanol for 24 hours at 4 "C. 3 parts LR White and 1 part 100% ethanol at 4 "C for 24 hours. Three changes in LR White at 4 "C for one day each.

The manufacturers of the LR White resin stress that it is essential that the polymerization temperature of 60 "C f 1 "C is strictly adhered to. However, we have a fairly good experience of curing LR White at 50 "C. It is essential that the polymerization takes place in the absence of oxygen. This can be accomplished by performing the polymerization in gelatine capsules that are filled up to the very top or under nitrogen. If you are used to a particular type of plastic for embedding, check whether it is compatible with LR White. Many plastics react with LR White resulting in an opaque reaction product that does not harden properly. Polymerize for one day and check whether the resin is hard enough for cutting afterwards. One should be able to just slightly deform the polymerized block at room temperature with one's fingernail. If not, continue the polymerization for another day. LR white cannot be kept for indefinite periods of time. Always store at 4 "C, squeeze half-empty bottles to keep the contact with oxygen to a minimum. If a block of polymerized resin forms in the bottle, do not use the resin. If the resin is too old, wrinkles will be seen in the sections, mostly in nucleoli. If this is seen, use a new batch of resin.

JI

2. Cut ultrathin sections and mount them on gold grids. Never use formvar coated grids (they do not withstand the in situ hybridization procedure). According to the size of the sections it is better to use meshed (150 or 200 mesh) gold grids or carbon coated gold grids. The grids should be thoroughly cleaned with ethanol and air-dried under clean conditions prior to use. It is helpful to consistently pull up the sections on one side of the grids (most grids display a bright and a dull side). In addition, it is necessary to dry the loaded grids thoroughly (e.g. overnight in an incubator at 37 "C) as LR White sections do not tolerate ethanol. Depending on your material and problem you might wish to consider ,,flat embedding" such as described by Steiner eta/. (1994).

6.4.3 Troubleshooting

JI

Block is too soft

troubleshooting

Check temperature of incubator Check for oxygen during polymerization Increase polymerization time Use a harder grade of resin

Block is not homogeneously hard If the outside of the block is softer than the inside, use a different type of plastic for embedding. If the surface is too soft, make sure no oxygen can contact the surface during polymerization If the inside is softer than the outside, the resin is too old and has to be discarded In Situ Hybridization at the Electron Microscopic Level

Block is too brittle Increase the number of changes in LR White and the duration of the changes

0

Sections float off the grids Clean grids using acetone. Cells appear to be not well penetrated, despite of long incubation in LR White The resin is too old and has to be discarded Sections break when exposed to the electron beam Use smaller sized meshes. Dry sections carefully. (Leave them in the EM column without turning on the beam for some time). Carefully check your dehydration protocol (excessivefragility may be caused by a high ethanol content in the resin). Sometimes it helps to move the beam over the bars lining a field of view of interest before viewing the section. Put the cells in the resin into vacuum before starting polymerization.

0

6.5 In Situ Hybridization at the EM level for DNA Sequences Both, biotin and digoxigenin labelled probes can be used for in situ hybridization at the EM level. We have found no difference between biotin and digoxigenin labelled probes. All of the steps described below can be conveniently performed in small block dishes on a shaker (mild shaking) at room temperature unless stated otherwise. Protocol

1.

Incubate the grids for 1 hour in RNAse A solution at 37 "C.

2. Wash 2x10 minutes in 2xSSC. DNA testing

3. Wash for 10 min. in TridCaCI2 buffer. Continue with steps 4/5 or 4a/5a. 4.

Incubate for approx. 10 minutes in proteinase K solution at

37 "C. 5. Wash 2x10 minutes in PBS/MgC12 buffer to stop the action of proteinase K. Continue with step 6. 4a. Treatment with pepsin solution as an alternative to treatment with proteinase K: Incubate grids for approx. 5 minutes in pepsin. 5a. Pepsin is stopped simply by immersion into lxPBS with p H at about 7.2-7.4. Continue with step 6.

6. F. WACHTLER et al

Fix the grids by incubation in paraformaldehyde for 10 minutes.

7.

Wash 2x10 minutes in PBS.

8. Wash 3x5 minutes in distilled water. 9. Place the grids onto a slide in such a way that they can be covered by a small cover slip (e.g. 18mm x 18mm). Blot away any water transferred with the grids with a filter paper. Immediately afterwards distribute the hybridization mixture over the grids (1OpI per cover slip is sufficient with up to 20 grids under each cover slip). Place the cover slip over the grids. It is important to ensure that there are no air bubbles left under the glass. The probe concentration has to be determined experimentally (concentrations which are too high, result in increased background labelling). 2-5ng/yl of probe is normally used for genomic probes, synthetic oligonucleotides can also be used at concentrations of 1-2ng/pl.

probe concentration

10. Put the loaded slides for 10 minutes in an incubator adjusted to

80 "C. 11. Transfer the slides into moist chambers (also heated to 80 "C) and these into an incubator adjusted to 37 "C in order to allow annealing of the probe to the target. This step should be carried out for a minimum of 4 hours. In practice, it is convenient to perform this step overnight. 12. Wash the grids 3x10 minutes in the stringency wash solution at 42 "C.

13. Wash the grids 2x10 minutes in 2xSSC at room temperature. The following steps are optional: 14. Incubate the grids for 10 minutes in RNase H buffer.

15. Incubate the grids for 1 hour with RNase H at 37 "C in a moist chamber (to reduce the amount of enzyme it might be advisory to put the grids on slides and cover the grids plus enzyme with a cover slip).

16. Wash the grids 2x10 minutes in 2xSSC. With this step the in situ hybridization procedure is finished. Next step is the immunocytochemical detection of the DNA-DNA hybrids, which will be treated below.

Hints RNAse A treatment removes RNA, thus avoiding the detection of DNA-RNA hybrids. The appropriate concentration of and/or incubation time in proteinuse K has to be determined for the specimen and structure in question. A balance has to be found between enhancement of probe access and sufficient maintenance of structure. A good alternative for proteinase K is pepsin. Pepsin seems to facilitate probe penetration very well. However, it may affect the ultrastructure of some organelles

choice of proteinase treatment

In Situ Hybridization at the Electron Microscopic Level

more than proteinase K. Generally speaking, EM sections are more resistant to proteolytic enzymes than light microscopic preparations. Both the time and concentration of pepsin may have to be adjusted experimentally. We denature both, target and probe DNA at the same time. It is, of course, possible to do this separately. 80 "C is a denaturation temperature approx. 10 degrees higher than the melting temperature (Tm) of the probe used.

denaturation temperature

stringency

F. WACHTLER et al.

Where n is the length of hybridizing sequence in base pairs (bp). Therefore the denaturation temperature of each probe can be calculated separately. 80 "C is a good general figure, however the temperature should never exceed 90 "C. If a higher melting temperature is required, then the concentration of formamide should be increased rather than the denaturation temperature. The denaturation temperature for EM in situ hybridization should be slightly higher than the Tm taking into account the inertness of the embedding medium. Again, a good balance between high yields of signal and well preserved ultrastructure has to be determined experimentally. It should be mentioned that there are plenty of alternative methods of denaturing the target DNA (e.g. N a O H ) . However, we found that using the protocol outlined above works well in a variety of applications, giving a good balance between ultrastructure and signal strength. The duration of renaturation is dependent on many factors (e.9. sequence complexity). As a rule of thumb: the higher the probe concentration and the shorter the length of the probe the shorter the renaturation period necessary. However longer periods of renaturation will do no harm. The temperature of the stringency washes should be adjusted for every probe (a more handy way is to vary the percentage of formamide) in order to obtain the optimal balance between a high concentration of specific label and low background. It mainly depends on the probe length and on the homology between probe and target. As a rule of thumb, the lowering of the stringency by 1 "C or 0.7% formamide allows for 1 % more base pair mismatch). It is recommended to perform this step in a heated shaking water bath. Optional treatment with RNAse H: RNase H specifically destroys RNA-DNA heteroduplexes. If RNA is still present, even after the RNase A treatment, it might cause background problems with the detection method. Therefore, RNase H treatment can further reduce non-specific DNA staining. However, in our hands it did not prove to be necessary.

6.6 In Situ Hybridization at the EM level for RNA Sequences In the following we outline a protocol for the detection of RNA using double stranded DNA probes. Those parts identical to the DNA-DNA hybridization protocol are not repeated in detail. For each target it has to be determined experimentally if a pretreatment (steps 1 to 5) is necessary. This mainly depends on the abundance and the accessibility of the target. For a high amount of target it is preferable to omit pretreatment and start with step 6, otherwise use the protocol as outlined below starting with step 1.

Protocol

1.

Wash the grids for 10 minutes in TrisKaCI2 buffer. (This step may be omitted.]

2.

Incubate the grids in either proteinase K or pepsin solution and stop the action of the enzymes appropriately. 2a. Optional incubation in saponin for 30 minutes to enhance target accessibility.

3.

Fix the grids by incubation in paraformaldehyde.

4.

Wash 2x10 minutes in PBS.

RNA

5. Wash 3x5 minutes in distilled water. 6.

Incubate the grids for 1 hour in prehybridization mixture at

42 "C. 7.

Denature the probe at 100 "C for 10 minutes and immediately quench on ice.

8.

Place the grids onto a slide in such a way that they can be covered by a small cover slip (e.g. 18 mm x 18 mm). Soak away the prehybridization mixture that transferred with the grids using filter paper. Immediately afterwards distribute the hybridization mixture over the grids. It is important that they do not dry out, but also that not too much prehybridization solution is left on them (to dilute the probe). Place a cover slip over the grids. It is important to ensure that no air bubbles are left under the glass.

9. Put the slides in an incubator at 42 "C in preheated moist chambers. This step should be carried out for at least 4 hours. In practice, this step is performed overnight. The temperature depends largely on probe length and homology of probe and target (rule of thumb: oligonucleotides up to 30 bp: 37 "C,genomic probes 42-45 "C) and has to be experimentally determined for each probe.

In Situ Hybridization at the Electron Microscopic Level

10. Wash the grids 2x15 minutes in 2xSSC at 42 "C followed by 1x15 minutes in 0.lxSSC at 65 "C. It is recommended to perform this step in a heated shaking water bath.

11. Wash the grids 2x10 minutes in 2xSSC at room temperature. The following steps of antibody detection are identical for RNA-DNA and DNA-DNA hybrids.

Hints In theory, one should take precautions to avoid degradation of target RNA. This means wearing gloves at all times, using sterile water treated with 0.1 % DEPC and using only sterile plastic material and glass baked at 200 "C for several hours. However, it seems that these precautions are not absolutely necessary for the detection of DNARNA hybrids.

6.7 Detection of Hybrids

detection systems

F. WACHTLER et al

In principle, any detection system using an electron dense compound can be used for the detection of hybrids following in situ hybridization at the EM level. The most commonly used are detection systems (such as antibodies) involving colloidal gold or the peroxidase reaction. Whereas colloidal gold gives a very precise localization of hybrids, protocols involving peroxidase may give results that are visually more informative. Another advantage of protocols based on the peroxidase reaction is, that the same protocol (and the same chemicals) can be used for light and electron microscopy. Furthermore, detection systems using peroxidase seem to be more sensitive than colloidal gold (see figs. 3, 4 and 5). Double labelling experiments can be easily accomplished by hybridizing simultaneously with a biotin-labelled and a digoxigeninlabelled sequence using two different detection systems (such as two different sized gold grains). In this case it is essential, that appropriate controls to exclude cross-reactions between the probes and/or detection systems are performed. In situ hybridization can be combined with immunodetection of other epitopes. The principle of this approach lies again in the utilization of two different markers for different antibodies. There are basically two approaches. If the epitopes do not survive the in situ hybridization protocol the immunostaining has to be performed before the hybridization and the grid fixed before the hybridization protocol. This will immobilize the colloidal gold on the grids. If the epitopes survive the hybridization protocol, the detection of the hybrids and the other epitopes can be performed simultaneously. In any case, the treatment with proteases has to be ommitted. In situ hybridization combined with immunodetection at the light microscope level is comprehensively explained in chapter 4.

In the following, we shall give a protocol both for the use of colloidal gold coupled antibodies as well as for the use of the peroxidase reaction.

6.7.1 Gold labelled Antibodies Protocol 1.

Following hybridization, incubate the grids in PBS supplemented with 1 % BSA for approx 30 minutes.

2.

Incubate the grids for at least 1 hour with the first antibody under moist conditions. This is usually done in drops of the solution on a paraffin film (at least $1 per drop if the grid is placed on top of the drop. It is, however, better to immerse the grid into a drop to enhance labelling. This requires at least 10 to 15p.l of antibody).

W

immune gold

3. Wash the grids 2x10 minutes in PBT pH 7.4. 4.

Wash the grids 10 minutes in PBT pH 8.2.

5.

Incubate the grids for at least 1 hour in the second, goldlabelled antibody under moist conditions. The antibody has to be diluted in PBT pH 8.2.

6.

Wash the grids 3x10 minutes in PBT pH 8.2.

7.

Wash the grids 3x5 minutes in deionized water (pH 8.2).

8.

Dry the grids on filter paper.

9. Stain the grids with aqueous uranyl acetate followed by lead citrate as for standard electron microscopy. Note that the slides must not be viewed in the electron microscope before the protocol is finished. Exposing the sections to the electron beam makes them inaccessible to antibodies and staining.

Hints Several books exist about immunocytochemistry at the EM level, therefore, only a few general considerations and hints are outlined in the following: Generally, it is better to use a two antibody- system (e.9. goat-antibiotin and rabbit-anti-goat conjugated with 5nm gold grains) in order to increase the .link" between DNA and gold grain thus enhancing the label density of the secondary antibodies. Three step systems do not seem to be superior to two step systems. For the protocol outlined above we use primary anti-biotin or antidigoxigenin labelled antibodies in a similar way. As secondary antibodies we use 5 nm colloidal gold coupled antibodies. Larger gold grains result in less intense label, smaller gold grains are difficult to see without silver enhancement. In Situ Hybridization at the Electron Microscopic Level

Fig. 3. Nucleolus of a CHO cell hybridized with a probe for the external transcribed b spacer RNA from the ribosomal gene repeat. The hybrids are visualized with 5 nm colloidal gold particles (arrows) Bar: 100 nm. It can be seen that the clusters of label overlay the dense fibrillar component and are not found outside the nucleolus. This means that the transcription and degradation of this type of RNA is located and confined to this particular component. Abbreviations: c...chromatin, d...dense fibrillar component, f...fibrillar center Fig. 4. Nucleolus of a human fibroblast hybridized with a probe for the human ribosomal gene repeat. The hybrids are visualized with 5 nm colloidal gold particles. Arrows point to label overlying chromatin, arrowheads point to label overlaying the dense fibrillar component. Bar: 100 nm It can be seen that most of the label is over the condensed nucleolus associated chromatin and only a small fraction overlies the dense fibrillar component. This is a typical situation for a cell with a low rate of rDNA transcription. Also note that the nucleolar components are not as easily identified as in conventional electron micrographs. Abbreviations: c...chromatin, d...dense fibrillar component, f...fibrillar center

The concentrations of each of the antibodies have to be determined experimentally.As a general rule, one should try to use the lowest possible concentration of the antibodies. Huge differences in the efficiencies of colloidal gold labelled antibodies exist between different batches produced by the same manufacturer and also between different manufacturers. If a detection protocol does not work, it is likely that the problem is with the secondary, gold labelled antibody. In principle avidin or streptavidin should be suitable for the detection of biotinylated hybrids. In our hands we found that anti-biotin antibodies give better results than avidin or streptavidin. Be careful with the use of lead citrate. Use it only when absolutely necessary. If the sections are incubated for too long, it may prove difficult to see the gold grains. To prevent loss of signal, make sure that the grids are completely dry prior to counterstaining.

6.7.2 Troubleshooting A previously successfully used antibody gives a low signal and/or a high non-specific background troubleshooting 0

0

0

0

F. WACHTLER eta/.

Vary the Tween-20 concentration. Varv the BSA concentration. Incubate the grids (prior to the first antibody) with the serum derived from the host of the secondary antibody. Add glycine (1 %) to the blocking solution. Check the antibody on other sections where it already successfully worked to exclude background by incomplete curing. If there is still high background label increase the NaCl content in the dilution of the secondary antibody up to 5 %. Use a new aliquot of antibody.

A new (batch or lot of) secondary antibody does not work in an established protocol Check the pH of all solutions that come in contact with the antibody, pH should not be below 8.0. Modify the antibody concentration. Try an antibody from a different manufacturer.

In Situ Hybridizationat the Electron Microscopic Level

A secondary antibody gives ,,clusters" of signal that may be nonspecific Spin down the antibody for 15 min. at 5000 g in an Eppendorf centrifuge, discard pellet. Use supernatant. Use a new aliquot of antibody.

6.7.3 The Use of the Peroxidase Reaction to Detect Hybrids at the EM level All steps are performed at room temperature unless stated otherwise.

Protocol

1. After the stringent washes (see above) the grids are washed in 4 x SSC for ten minutes.

@ -

peroxidase stain

z. Incubate the grids for 20 minutes at room temperature in 4xSSC containing 5 % non fat dried milk and 0.05% Tween 20. Incubate the slides for between 30 minutes and two hours in 1 mg/ml Avidin-peroxidase dissolved in 4xSSC containing 5 % non fat dried milk and 0.05 % Tween-20. (To keep the volume of liquid small, this step can conveniently be performed by placing the grids between a slide and a coverslip in a moist chamber. It is a good idea to check under phase contrast for any trapped air bubbles). Do not use a shaker. 4. Wash the grids 3 times for ten minutes each in 4x SSC containing 0.05 % Tween-20.

A

use fume cupboard

5. Incubate the grids in a mixture containing 10 m M imidazole, 50 m M Tris HCI, 0.05 % diaminobenzidine for 20 min. at 4 "C. Filter this solution before use, using a fine mesh filter such as a Millipore filter.

6 . Incubate the grids in a mixture containing lOmM imidazole, 50mM Tris HCI, 0.045 % H202 and 0.05 % diaminobenzidine for 20 minutes at 4 "C. (It is a good idea to monitor this reaction by stopping individual grids at five minutes intervals).

7. Stop the reaction by transferring the grids into five changes of distilled water at 4 "C. 8. Air dry. Hints Blocking endogenous peroxidases in the nucleus seems to be unnecessary as the signal/noise ratio is unaffected by such treatment. However, it may be necessary to block peroxidase in the cytoplasm of cells with H202. DAB can be prepared and stored at -20 "C as a 1 % stock solution. Dissolving of DAB is facilitated by first preparing a 10% solution in DMSO. Dissolving should be performed in the dark. F. WACHTLER et a/.

6.7.4 Troubleshooting The hybridization does not work in spite of a well labelled probe De'crease the ratio of labelled dUfP/dTTP Increase the denaturation temperature Decrease the temperature of the stringent washes

troubleshooting

High, apparently non-specific background Use another batch of antibodies. Perform a prehybridization step (i. e. perform a denaturation, renaturation step with a hybridization mixture that does not contain labelled probe). Increase the temperature for denaturation. Decrease the concentration of the antibodies. Increase the temperature or the formamide concentration for stringent washes. low, but probably specific signal Increase the unlabelled ,,carrierN nucleic acids in the hybridization mix. Decrease the glutaraldehyde concentration in the fixative. Increase the duration of the proteinase treatment.

Fig. 5. Nucleus of a human lymphocyte from peripheral blood hybridized with a probe for the transcribed part of the human rRNA gene repeat. The hybrids are visualized with the peroxidase-DAB reaction. Note the the ring-shaped signal overlying the nucleolus (arrow) and a second cluster of label (arrowhead) in the condensed chromatin, reflecting non-transcribed rDNA. Bar: 100 nm In Situ Hybridization at the Electron Microscopic Level

Use smaller sized gold grains. Decrease the temperature or the formamide concentration for the stringent washes.

6.7.5 Controls

controls

F. WACHTLER et of.

The general rule, that one should perform as many controls as one can think of, is particularly true for in situ hybridization at the EM level. This is particularly important when implementing a new protocol or modifying an established procedure. According to our experience, it is very helpful to supplement and corroborate in situ hybridizations at the EM level with parallel experiments on the same material at the light microscopic level, whenever this is possible. Hybridization on the light microscopic level has been covered extensively elsewhere in the book and so it will not be dealt with here in detail. Examples of possible control experiments follow (see Fig. 6a and 6b). Light microscopy on chromosome preparations : This procedure is useful, if a DNA probe for a known chromosomal locus is used, because it will show, whether the probe hybridizes specifically to the chromosomal locus or not. In addition, by comparing metaphase and interphase cells, it will become apparent, if accessibility problems exist. Furthermore, this control is helpful to check, whether a protocol intended to detect RNA only detects DNA or not, since there should be no label over metaphase chromosomes. DNA halo preparations as targets for in situ hybridizations : If very specific information on the hybridization pattern is required, DNA halo preparation may be used as targets for hybridizations. The principle of DNA halo preparations is to release loops of DNA from nuclei. In these loops single, fully extended fibers of DNA can be seen. Using DNA halo preparations as targets for in situ hybridization one can directly visualize the formation of single DNA hybrids and can thus iudge, whether the probe used detects a specific DNA sequence as expected. Semi-thin sections of embedded material : This control is particularly useful, if all the above controls gave a positive result, but the in situ hybridization at the EM level failed and one is to determine, whether the embedding or the detection system are faulty. It can be performed using the same protocol as the one given for EM grids. If one uses different detection systems, such as fluorochrome labelled antibodies, not peroxidase, it is strongly recommended to check for possible autofluorescence of the resin and of the cells. Controls with gold-coupled antibodies: According to our experience, most problems are caused by the gold labelled antibodies. It is a good idea to develop an antibody test system as follows: One should obtain an antibody directed against an abundant epitope in one’s material (e.g. actin or an intermediate filament protein) from the same species as the antibodies one uses for the detection of digoxigenin or biotin. Using this antibody, one can check the staining pattern of the gold labelled antibody used for in situ hybridization.

Fig. 6a. Nucleolus of a human Sertoli cell hybridized with a probe for rDNA. The hybrids are visualized by fluorescence. Bar: 5 pm Fig 6b. This shows the same at the EM level visualized with 5 nm gold grains Arrowheads point to label over the dense fibrillar component. Bar: 500 nm Note the ,,8”-shaped fluorescence signal in fig 6a that corresponds to the (labelled) dense fibrillar component at the EM level. Abbreviations: c...chromatin, d...dense fibrillar component, f...fibrillar center, g...granular component

Fig. 7. Metaphase and interphase from a culture of human PHA-stimulated lymphocytes hybridized with a probe for human rDNA. Small arrows point to the label at the NORs, a large arrow points to the rDNA in the interphase nucleolus. Bar:

10 pm

The hybrids, visualized by fluorescence, can be seen in the known chromosomal locations: the nucleolar organiser regions (NORs)on the short arms of the ten acrocentric chromosomes

In Situ Hybridization at the Electron Microscopic Level

Fig. 8a. Metaphase and interphase nuclei from a culture of human PHA-stimulated lymphocytes stained with DAPl to visualize DNA Fig. 8b. Same field of view as in fig 8a, hybridized with a probe for human rRNA. Hybrids are visualized by fluorescence. Arrows points to the rRNA in the interphase nucleoli. Note the absence of label at the chromosomal NORs. Bar: 10 pm

F. WACHTLER et al.

Fig. 9. DNA halo preparation hybridized with the 7.5 and 8.1 Kb fragments of the human ribosomal RNA gene repeat, visualized by fluorescence. One sees a periodic repeating pattern of two adjacent fluorescent lines (arrows). Bar: 10 pm Since the 7.2 and 8.1 fragments are separated by 7.3 and 19.5Kb respectively, it can be seen that the probe hybridizes as expected

Fig. 10. Semithin section of resin embedded human PHA-stimulated lymphocytes hybridized with a probe for rDNA, visualized by fluorescence. Arrows point to hybridization signals in nuclei. Bar: 10 pm

In Situ Hybridizationat the Electron Microscopic Level

Antibodies, especially gold-labelled antibodies tend to bind nonspecifically to sections in an unpredictable way. It is therefore essential that the following negative controls are performed:

1. In situ hybridization protocol omitting labelled probe from the hybridization mixture. 2. In situ hybridization protocol omitting the denaturation step.

6.8 Evaluation and Interpretation of Results The general principle of evaluation is to determine the location and density of the electron dense marker used to detect the labelled hybrid nucleic acid sequences. In the case of the peroxidase reaction, this is done by visual inspection. In the case of gold grains, visual inspection generally is not as intuitively informative on one hand, on the other hand the discrete and particulate nature of gold grains allows a quantitative evaluation. We feel, that this quantitative evaluation should not be pushed too far and used only to corroborate qualitative statements. The principle of the data analysis using colloidal gold grains to visualize hybrids is to determine the number of grains within a particular cellular compartment. The simplest way of doing so is by counting gold grains and correlating these numbers to the areas of cellular components. Areas can be determined by a variety of methods, for instance by using a point mask or a PC based measurement program such as Sigmascan (Jandel Scientific, Sausalito, Ca.). Normally, this is done on the basis of several micrographs. For easy evaluation all micrographs should be taken at the same magnification and magnified during printing to the same final magnification. Some electron microscopes equipped with an integrated image analysis system (such as ,,PC- Image", Foster Findlay, Newcastle upon Tyne) allow the quantitative analysis on the basis of directly recorded digital images. The determination of label density within a compartment (a volume) is facilitated by the basic stereological assumption that within random sections through cells, the relative volumes are proportional to the relative areas within such sections. Thus, in order to determine the signal density within a particular compartment (volume) of a cell, it is sufficient to determine the number of gold grains per area of the sections of the said compartment. If gold coupled antibodies are used to visualize a signal some considerations need to be taken into account:

1. Correlation between the site of a gold grain and the site of the nucleic acid sequence to be detected. The length of an antibody molecule is approximately 12 nm, thus if two antibodies are used to detect a labelled DNA hybrid, the position of the hybrid molecule can be estimated to be within 25 nm of the seen gold particle. F . WACHTLER et 01.

Fig. lla. Electron micrograph of a human Sertoli cell, the DNA of which has been visualized by colloidal gold Fig. 11b. Area determination using a morphometry program (Sigmascan, Jandel) of the cell in Fig. 11a (The areas in cm as displayed on the screen have to be divided by a factor of approx. 1.4 due to the reduction in size of the micrograph)

2. Signal to background ratio. Gold labelled antibodies tend to have a high background. Therefore the background label density of gold grains needs to be determined for all experiments and taken into account in the evaluation and interpretation of results. Normally one would determine the background level of signal by counting the gold grains over an area of resin not containing cells and over cellular compartments, where no specific level is expected to be found. In Situ Hybridization at the Electron Microscopic Level

3. Statistical analysis of data. An important point for the interpretation of results is the determination of a background value as a value to which a supposedly positive signal density over a component is to be compared. The obtained data for a signal density value should be analyzed whether they are distributed according to a Gaussian distribution. If so, Student's t- test may be used, if not, a non- parametric statistical test such as the chi- square test or the Kolmogoroff- Smirnoff- test should be performed. This occurs especially, if a nucleic acid sequence is not distributed homogeneously within a cellular compartment, which is often the case. 4. One should always be aware of the fact that one visualizes only a minute fraction of a nuclear acid sequence on an EM section. As suggestive as a single micrograph may be, never rely on it without other corroboration!

Acknowledgements We are grateful to Dr. Barbara Sollner-Webb for making the external transcribed spacer RNA from the ribosomal gene repeat (Fig. 3) probe available to us.

References Steiner, M., C. Schaefer, and W. Mosgoeller (1994) In situ flat embedding of monolayers and cell relocation in the acrylic resin LR White for comparative light and electron microscopic studies. Histochemical Journal, 26, 934-938. Thiry, M., and L. Thiry- Blaise (1989) In situ hybridization at the electron microscopic level: an improved method for the precise localization of ribosomal DNA and RNA. Eurp. J. Cell Biol., 50, 235-243. Thiry, M., and L. Thiry- Blaise (1991) Locating transcribed and non- transcribed rDNA spacer sequences within the nucleolus by in situ hybridization and immunoelectron microscopy. Nucleic Acids Research, 19, 11-15. Wachtler, F., C. Schoefer, W. Mosgoeller, K. Weipoltshammer, H.G. Schwarzacher, A. Stahl, I. Gonzales, and J. Sylvester (1992) Human ribosomal RNA gene repeats are localized in the dense fibrillar component of nucleoli. Light- and electron microscopic in situ hybridization in human Sertoli cells. Exp. Cell Res., 198, 135-143. Wachtler, F., and A. Stahl (1993)The Nucleolus: A functional interpretation. Micron, 24,473-505.

F. WACHTLER et al.

In Situ Hybridization Melody Clark Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Appendix I

Labelling DNA Probes for In Situ Hybridizations

Whilst the number of uses and methods for in situ hybridization continually increase, the number of probe labelling protocols remains relatively constant eg. nick translation, random priming, PCR, oligonucleotide synthesis, end labelling etc. (for a collection of protocols see Boehringer, 1993). The vast majority of the chapters in this manual use DNA probes. So to avoid repetition, labelling protocols used by each of the authors have been combined in this appendix. For recommendations of which to use for particular in situ hybridization techniques, consult individual chapters. The labelling of RNA probes is described separately in Chapter 5 : In situ hybridization to embryo whole mounts and tissues sections. The alternative is to buy in ready-made probes from a commercial source. Whilst this may be a viable option for certain situations eg. human chromosome painting, these are not always available and are also relatively expensive. The methods which will be described here are:

Nick translation PCR Random hexamer Suppliers are not listed, but labelling involves standard molecular biology chemicals which are available from any of the major manufacturers, such as Boehringer Mannheim, Sigma and Gibco-BRL. Suppliers of labelled nucleotides can be found in the chemical listings of each chapter as the different authors have their own individual preferences and proven methods for either biotin, digoxigenin or fluorescent probes. Fluorochrome-labelled probes should be stable for few months at -20 "Cin a light protected container, biotin- or digoxigenin-labelled probes are stable for a much longer time.

M. Clark (Ed.), In Situ Hybridization. 0 Chapman & Hall, Weinheim. ISBN 3-8261-0038-7

Nick Translation Supplied by K. Anamthawat-Jonsson

This is based on the method of Rigby et al (1976). The protocol described here can be used with fluorochrome-, digoxigenin-, or biotin-coniugated nucleotides and is suitable for labelling: Total genomic DNA Insert excised from a plasmid digest Total plasmid DNA containing both vector and insert. Sequences which are too big to efficiently amplify using PCR The method involves nick translation of a double stranded DNA template by pancreatic DNase-l and E. coli DNA polymerase-I, followed by an ethanol precipitation of the labelled total genomic DNA together with unlabelled salmon sperm carrier DNA.

Solutions RNase free DNA template labelled nucleotide: Fluorochrome label (1 mM stock) eg. fluorescein-11-dUTP (green), rhodamine-4-dUTP (red), or coumarin-4-dUTP (blue); digoxigenin-lldUTP or biotin-16-dUTP (1 mM stock).

10 x reaction buffer: 0.5 M Tris HCI (pH 6.8),50 mM MgCI2; 0.5 mg/ml BSA 10 x unlabelled nucleotide mixture: 0.5 mM of dATP, 0.5 mM dGTP ,0.5 mM dCTP in lOOmM Tris-HCI, pH 7.5 Unlabelled dTTP: 0.5 mM solution in lOOmM Tris-HCI, pH Z5 100 mM DTT

10 x labelling enzyme mix: 4Opg/pl DNase-l and 0.4U/pl DNA polymerase-I

0.3 M EDTA, pH 8. Salmon sperm carrier DNA (sheared) (5mg/ml) Ethanol precipitation chemicals: 3M Sodium acetate, 70 % ethanol, Absolute ethanol TE buffer: 10 mM Tris-HCI, 1 mM EDTA, pH 8.

Equipment Cooled waterbath at 14-15 "C Vacuum desiccator Microfuge

K. ANAMTHAWAT-JONSSON

Method Pretreatment of DNA (only required if total genomic DNA is used): Prior to labelling, RNase-free total genomic DNA is sheared to 3-10 kb lengths by sonication or mechanically by drawing the DNA solution about 100 times through an ultrafine hypodermic syringe.

nick translation labelling

1. Mix together on ice :

Add sterile distilled water to make volume up to 45pl. Mix all the components well and centrifuge briefly, then add 5pl 10 x labelling enzyme mix. Mix gently and incubate the reaction at 15 "C for 1.5 h (digoxigenin, biotin) or 3 h (fluorochrome).

2. Add 51.11 of 0.3M EDTA to stop the reaction, followed by 2pl of salmon sperm carrier DNA.

3. Immediately precipitate DNA by adding 1/10 volume of 3M Sodium acetate and 3 volumes of ice cold ethanol. Keep at -20 "Cfor 1 hour or 30 minutes at -70 "C. Spin down in a microfuge at 12,000 rpm for 15 minutes. 4. Discard the supernatant and wash the pellet gently with 70% ethanol. Dry the pellet briefly in a vacuum desiccator.

5. Resuspend the DNA pellet in an appropriate volume of TE buffer. Consult individual protocols for guidelines as to final probe concentration.

6. Take 1 pI of the labelled probe and blot onto a nylon or nitrocellulose membrane. Detect the rate of incorporation of the labelled probe and of a positive and negative control using the appropriate detection system (see Appendix 11).

Notes

1. Nick-translation kits can be purchased commercially. If many labelling experiments are to be carried out, they do work out more expensive than buying the individual components separately. However they do have the advantage of being optimised and contain test DNA for troubleshooting purposes. Do note that some commercial kits may contain pre-made unlabelled nucleotide mix for use with only one labelled nucleotide, usually dCTP Check carefully before ordering. Labelling DNA Probes for In Situ Hybridizations

2. The labelling enzymes can also be bought in a ready-made mix from Gibco BRL. Use as a lox stock solution. 3. Labelling whole plasmids, as opposed to excising the inserts and labelling these separately can result in more efficient probes (due to network formation during the hybridization reaction).

4. The labelled DNA is fragmented by the nick translation procedure. check length of fr 3

*h-"prjlxi*llr

rlJ@lr"*&*%?Nr

The length of the fragments should be in the order of magnitude of 300 to 500bp (this can be checked by running some of the labelled DNA out on an agarose mini-gel with appropriate size markers eg. 123bp ladder). Longer fragments can be shortened either by sonication, increasing the DNAse I concentration or leaving the reaction for longer.

5. A cheap alternative for a cooled water bath is a styropore (polystyrene) box or a household vacuum flask filled with iced water). PCR machines can also be used.

Probe Labelling and Amplification by PCR Supplied by C. Schofer This method is adapted from Lo et al (1990)and has the advantages of high labelling incorporation and amplification of final product. This protocol outlines the labelling and amplification of inserts in plasmids. Biotin dUTP is used below, but fluorochrome- and digoxigeninconjugated nucleotides can also be incorporated sucessfully. This protocol is suitable for labelling: Inserts in plasmids Single copy sequences from genomic DNA

Solutions 20x reaction A x : 1M KCI/O.OSM MgCI2/0.2MTris (pH 8.3)/0.2% gelatin Unlabelled nucleotide mix 20mM each of dATP, dCTP, dGTP and 15mM dTTP

0.3mM biotin-dUTP 100 pM primer 1 100 pM primer 2 Taq DNA polymerase

Equipment

PCR machine agarose gel electrophoresis equipment C. Schofer

Method

1. Mix together:

PCR labelling 8t amplification

2. 5U Taq DNA polymerase The final reaction volume should be made up to lOOpl with SDW. Overlay the PCR mixture with 7Opl mineral oil

3. Run the following programme: 92 "C for 2 minutes 92 "C for 30 seconds 55 "C for 30 seconds 72 "C for 30 seconds Repeat last three stages for 30 cycles Run a final extension step of 72 "C for 5 minutes 4. After removal of mineral oil, run 5pI of the reaction on an agarose mini-gel to check for efficient production of probe and noncontamination. In the case of the fluorescein probes, the pellet is yellow and the unincorporated fluorescein-UTP runs at the front during electrophoresis and is easily seen on the UV-transilluminator. The DNA can then be ethanol precipitated and resuspended in TE buffer.

Notes One should always check the amplified DNA with DNA gel electrophoresis. Only a single band should be obtained. Any other bands indicate non-specific priming and result in contaminated probes which may give false results. It is also a good idea to include an extra negative control, where the same reaction is set up, but the dTTP concentration is increased to 20mM in the l o x unlabelled nucleotide and the labelled nucleotide and DNA template are left out. N o bands should appear when this sample is run on a gel. If bands are present contamination of the PCR reaction has occured, in which case start with fresh reagents.

e\ check priming specifity

2. lox reaction buffer is often supplied with individual Taq polymerase enzymes. The concentration of magnesium chloride varies between maufacturers and it is important to check that this is consistent with your reaction conditions.

Labelling DNA Probes for In Situ Hybridizations

3. The use of small amounts of template DNA (eg. 5-long) prevents non-specific priming and amplification of plasmid DNA. test optimum annealing temperature

4. Note that different primers require different annealing temperatures. 55 "C, given here is the standard temperature for M13 based primers. To calculate the approximate annealing temperature of a primer, count 4 "C for each G/C base and 2 "C for each A/T base.

5. The vast majority of labs, clone into pUC based vectors, eg. Bluescript. In this case, M13 forward and reverse, KS/SK and T7/T3 primers will sucessfully amplify the clone. But always check beforehand. Maps of the most common plasmids can be found in the back of most chemical company catalogues, along with listings of suitable primers. 6. The extension time at 72 "Ccan be altered according to the length of the sequence to be amplified. Theoretically Taq DNA polymerase can amplify a maximum of 3.6Kb per minute, but a safer and more practical figure is to use half this ie. 1.8Kb per minute. A 30 second extension time is adequate for most PCR reactions.

optimum label content

7. Note, that in this protocol, the ratio of biotin-dUTP to dTTP is 1 :3. When using digoxigenin labelled dUTP, the ratio can be reduced to 1 : 10. There is an upper limit to the incorporation of labelled nucleotides. This limit is not caused by PCR but by in situ hybridization. Too intensively labelled probes result in poor hybridization efficiency.

8. Probes are generally amplified from clones in plasmids, however it is possible to amplify directly from genomic DNA or cDNA if sequence data is available. It is important in this case that only a single band is obtained. The presence of several bands indicates non-specific priming of other unknown sequences, resulting in an impure probe.

9. To check the incorporation of the labelled nucleotides a dot blot should be performed.

Random Hexamer Primed DNA Synthesis Supplied by G. Evans With this method is based on Feinberg and Vogelstein (1983;1984). The klenow fragment of DNA polymerase I utilises random oligonucleotides as primers for copying single-stranded templates incorporating labelled nucleotides at high efficiency. It only requires small amounts of DNA eg. 25ng and is also fairly tolerant of impurities in the DNA sample. This method is generally used for the same situations as nicktranslation. Which method is used, is purely down to personal preference. G . Evans

Solutions 5x labelling buffer: 3.35M

HEPES/0.85M Tris-HCV85mM MgCI2/165mM

P-mercaptoethanol pH 6.8 5x nucleotide mix: 0.1 m M each of dATP, dGTP, dTTP and biotin-dCTF! 5x BSA: 6.65mg/ml 5x oligodeoxyribonucleotide primers (hexamers) 1 mg/ml Klenow fragment of DNA polymerase at 5U/pI

0.2M EDTA

Method 1. Denature the DNA template at 95 "C for 5 minutes, quench on ice.

W

random hexamer labelling

2. Add to this to l x final concentration, the 5x labelling mix, 5x nucleotide mix and 5x BSA with 50ng-1 pg of DNA. The final volume should be 501.11. Add 1 pl of Klenow enzyme. 3. Incubate at room temperature for 1 hour. 4. Terminate the reaction by adding 5pl of 0.2M EDTA.

5. The probe can be used directly without further purification. Notes

1. Random hexamer kits can be purchased commercially, but it is often cheaper to buy the individual components and make your own buffers. However kits do have the advantage of being optimised and contain test DNA for troubleshooting purposes. Do note that some commercial kits may contain pre-made unlabelled nucleotide mix for use with only one labelled nucleotide, usually dCTP Check carefully before ordering.

2. Random hexamer labelling is usually more tolerant of impurities in a DNA sample than nick translation. It should certainly be the preferred method for labelling DNA fragments directly from agarose gels. It also does not require so much DNA template, as nick translation.

3. To check the incorporation of the labelled nucleotides a dot blot should be performed.

Labelling DNA Probes for In Sifu Hybridizations

Probe Labelling: Generalised Troubleshooting Probe is not labelled well: Test label a control DNA sample (either one supplied with a kit, or a previous sample which is known to work): Control does not label: Labelling enzyme inactivation : Generally these labelling methods are quite tolerant when it comes to purity of DNA. The usual problem with labelling is that the labelling enzyme has become inactivated.This is particularly a problem with the DNaseVDNA polymerase I mixture for nick translation as both enzymes are relatively heat labile, functioning most efficiently at 15 "C. With PCR, it is generally the lox dNTP mix which needs replacing, precipitating a fresh set of primers may also help. Control labels: Template problems : The control reaction means that the reaction mix and enzyme are effective, so possible causes and solutions include:

Dirty template: repurify or try a different labelling method. Modify the ratio between labelled dUTP and dTTP Use labelled dATP instead of labelled dUTP

References Boehringer Mannheim (1993)The DIG system user's guide for filter hybridization. Boehringer Mannheim p 1-90. Feinberg, A.P; Vogelstein, B (1983)A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13. Feinberg, A.P; Vogelstein, B (1984) Addendum : A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137, 266-267. Lo, Y.M.D; Mehal, W.Z; Fleming, K.A. (1990) Incorporation of biotinylated dUTP, in D.H. Gelfand, J.J. Sninsky and T.J. White (eds) : PCR protocols. A guide to methods and applications. p 113-118. Academic press, Sun Diego, NY. Rigby, PW.J; Dieckmann, M; Rhodes, C; Berg, P (1977).Labelling deoxyribonucleic acid to a high specific activity in vitro by nick translation with DNA Polymerase-I. J. Mol. Biol. 113, 237-251.

G. Evans

In Situ Hybridization Melody Clark Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Appendix II

Quantification of Efficiency of Incorporation of Labelled Nucleotides Supplied by F. WACHTLER ef al.

Incorporation of the labelled nucleotide should always be verified by a dot-blot analysis against an appropriate standard. The standard can either be obtained commercially or made “in house”. The basic procedure involves blotting newly labelled DNA onto a membrane alongside a verified standard and performing an antibody detection of the labelled nucleotides. The level of incorporation is roughly quantified by comparing the intensity of the colour reaction of the newly labelled probe with the standard. Two protocols are described here, but these can be varied extensively (e.g. by using differently labelled antibodies, or by using another detection system such as chemiluminescence etc.).

Establishing a Control Sample

A control sample is useful for adopting the protocol in a new lab and for use as reference to further labelling. Digoxigenin- or biotin- labelled plasmid can be obtained from suppliers of the nucleotides, or should be made. Fluorochrome-labelled control can be made using wheat total genomic DNA (commercially available, or isolated from fresh leaves) and nick translating with 30pM rhodamine-4-dUTP and

15pM dTTf?

Preparing the Blot Blot 1 p1 of the test DNA, a positive and a negative control onto a nylon membrane. Quantification may be refined by blotting a dilution series of the positive control alongside the test DNA. Immobilize the DNA by baking at 120 “C for 30 minutes or under a UV-lamp for 3 minutes. (Use small parts of membrane : a few square cm will do - in order to keep the amount of solutions low). Perform either of the following two detection rections, depending on the label incorporated into the probe. NB: All steps are carried out at room temperature (unless stated otherwise) in a Petri dish on a shaker. Volumes of solutions required are indicated in brackets. M. Clark (Ed.),In Situ Hybridization.0 Chapman & Hall, Weinheim. ISBN 3-8261-0038-7

Protocol for the Determination of Biotin Incorporation Solutions 2xSSC: 300mM NaCl + 30mM sodium citrate (50ml). "milk": 5 % non-fat dry milk in 2xSSC (20ml). avidin/POX: dilute avidin-POX complex to a final concentration of 1pg/ml in 2xSSC (10ml).

A use fume cupboard

DAB: Dissolve DAB in TrisHCl/imidazole buffer (50mM TrisHCl + lOmM imidazole) to a final concentration of 0.05% (filter the solution, if the DAB is not completely

solved. (20ml).

Developing solution: add H202to a final concentration of 0.05% to the DAB (20ml). Use fresh H202.H 2 0 2can be stored at a concentration of 30% at 4 "C for some months.

Method

1. Rehydrate the membrane in 2xSSC for 1 minute.

2. Incubate the membrane for 30 minutes in "milk" in order to preassay of biotin incorporation

vent non-specific binding of avidin.

3. Incubate the membrane for 30 minutes in diluted avidin- peroxidase (avidin-POX) complex.

4. Wash 2x15 minutes in 2xSSC. 5. Incubate the membrane for 5 minutes in the DAB solution in the dark.

6. Incubate the membrane in DAB/H202solution in the dark. The DAB acts as a substrate for the peroxidase. The incubation will result in a brown precipitate. The time of incubation should be checked visually (it can take between a few seconds to 30 minutes).

Z Stop the reaction by immersion of the membrane in ethanol. The membrane can be dried and stored.

Protocol for the Determination of Digoxigenin Incorporation The basic stages in the detection of incorporation of digoxigenin is essentially the same as the detection of biotin. The following protocol is recommended by Boehringer- Mannheim (DIG labelling and detection kit):

Solutions The volumes of solutions in brackets indicate the amount needed for one experiment. However, buffers 1 to 4 can be prepared in larger quantities and stored. F. WACHTLER et a1

Buffer 1: 150 mM NaCl + 100 rnMTrisHCl + 7 % Sodium dodecyl sulfate (SDS) (pH 7.5). (50ml). Buffer 2: 1 % blocking reagent in buffer 1 (aliquots of a 10% stock solution can be stored frozen). (20ml). Buffer 3: 100 mM NaCl + 100 mM TrisHCl + 50 mM MgCI2 (pH 9.5). (30rnl). Buffer 4: lOOmM TrisHCl + 1mM EDTA (pH 8.0). (10ml) Antibody: Dilute Alkaline phosphatase-coupledanti-digoxigenin antibody 1 5000 in buffer 2 (10ml). Developing solution: 450pg/rnl NBT and 175pg/rnl BClP in buffer 3 (10ml).

A

use fume cupboard

Method

1. Incubate the membrane for 1 minute in Buffer 1. 2. Incubate the membrane for 30 minutes in Buffer 2. 3. Incubate the membrane for 30 minutes with the diluted antibody.

4. Wash the membrane 2x15 minutes in Buffer 1. 5. Incubate the membrane for 2 minutes in Buffer 3.

assay of digoxigenin incorporation

6. Incubate the membrane in the developing solution in the dark. This solution acts as substrate for the AF! Monitor the progress of the development visually. The reaction leads to a deep blue precipitate.

7. Stop the reaction with Buffer 4. The membrane can be dried and stored. The colour turns pale on dry membranes but can be refreshed by incubation in Buffer 4. Alternatively, the membrane can be rinsed in ethanol which makes the staining reaction more permanent.

Notes

1. These reactions can be obtained in kit form, but obviously, the cost is much greater than if the chemicals are bought separately.

2. No suppliers are listed, but all of the chemicals can be obtained from the maior manufacturers, in particular, Sigma, Vector laboratories and Boehringer Mannheim.

Troubleshooting

If the probe does not label well, compared to the standard, check the troubleshooting guide in Appendix I. If the standard DNA also fails to be visualised, something has become inactivated or contaminated in the detection chemicals : start again with fresh chemicals. In particular check pH values and for precipitation of the developing chemicals. Quantification of Efficiency of Incorporation of Labelled Nucleotides

In Situ Hybridization

Glossary

Melody Clark Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Allele: (as in text: allelic loss). One of two or more forms of a gene occupying the same locus on a particular chromosome. Allopolyploid: (as in text: Allopolyploidization). An individual which arises (either spontaneously or through experimental induction) from the crossing of two or more species or genera and contains the structurally and genetically different whole chromosome sets of the parents. Thus representing an increase in number of chromosome sets over each of the original parents. Aneuploid: Cell or individual in which one or more whole chromosomes are absent from or additional to a euploid chromosome complement e.g. man : 2n = 2x-1 = 45 or 2n = 2x+1 = 47. Centromere: Also called the primary constriction. Region of the chromosome with which spindle fibres associate during mitosis and meiosis. Constitutional mosaicism: Presence within an individual of cells differing with respect to their chromosome structure or chromosome number. e.g. In man a Turners mosaic individual can have a mixture of cells with 2n=2x-1 = 45 XO and 2n=2x = 46 XX, the proportion of which is individual and tissue specific. Cosmid: Cloning vector used to clone fragments of between

35-45Kb. The vector is a modified plasmid which carries a copy of the DNA sequences (cos sequences) required for packaging DNA into bacteriophage lambda particles. They also carry an origin of replication and a drug resistance marker so that they can also be transformed and propagated as plasmids. Cot- 1 DNA: Highly repetitious and moderately repetitive fraction of genomic DNA. Crossing over: Physical events involving the reciprocal exchange of segments at corresponding positions along pairs of chromosomes by symmetric breakage and reunion leading to genetic recombination between linked markers. M. Clark (Ed.),In Situ Hybridization. 0 Chapman & Hall, Weinheim. ISBN 3-8261-0038-7

Dysplasia: (as in text: dysplastic alterations). Abnormal development

of an organ or part of the body. Granulopoietic precursor cells: White cell precursors. Hapten: Incomplete antigen that can stimulate antibody production only when complexed to a particular protein. In text this refers to digoxygenin and biotin which require a second reporter molecule to reveal sites of hybridization. Hematopoietic system: Bone marrow system. Homoeologous chromosome: Partially homologous chromosome. Homoeology designates residual homology between modern chromosomes which evolved from a common ancestral chromosome. Homologous chromosome: Chromosome or chromosome segments identical with respect to structure and constituent genetic loci. Karyotype: Chromosome complement of an individual or group of individuals defined by the number and morphology of the chromosomes at mitotic metaphase. Marker chromosome: Novel chromosome or structural change associated with a disease state or cell line. eg. In man chronic myeloid leukaemia is associated with the Philadelphia chromosome, seen as a smaller chromosome 22 as a result of an unequal translocation between chromosomes 22 and 9. Micronuclei: Nucleus separate from and additional to the main nucleus of a cell. This is produced during the telophase of meiosis or mitosis by lagging chromosomes or chromosome fragments derived from spontaneous or experimentally induced chromosome changes. Monosomic: Aneuploid cell, tissue or individual with one chromosome missing from an otherwise normal diploid chromosome complement. e.g. In man : 2n=2x-1 = 45. Neoplasm: New abnormal growth of tissue ie. a tumour.

NOR (Nucleolar Organiser Region): Also called the secondary constriction. Region of the chromosome active in nucleolus formation and the site of ribosomal RNA genes. Pachytene chromosomes: Stage of meiosis where homologues are paired, but not very constricted in size compared to metaphase: source of elongated chromosomes for physical mapping. Pathognomic: Characteristic or indicative of a particular disease. Glossary

Polyploidization: Conversion (either spontaneous or experimentally induced) of cells or individuals from the haploid or diploid state to a higher number of complete chromosome sets. eg a tetraploid has four complete chromosome sets (2n=4x). Recombination: Set of processes [involving crossing over) that results in new linkage relationships of genes, giving rise to chromosomes carrying genes derived from different parents. Retrotransposon: Type of transposon which moves by producing an RNA copy which is then reverse transcribed and integrated into the genome as a DNA copy. e.g. LINES and SINES sequences in humans. Robertsonian translocation: Chromosome structural change due to centric fission [production of two acro or telocentric chromosomes from one metacentric) or centric fusion (fusion by reciprocal translocation of two acrocentric chromosomes to give one metacentric chromosome). Satellite DNA: Denotes any DNA of sufficiently different composition to the maior DNA component to separate as a distinct band during isopynic caesium chloride centrifugation. Usually a family of highly repetitious repeat sequences, often localized at centromeres. Southern blot: DNA fragments separated according to size by electrophoresis through an agarose gel. The DNA is denatured within the gel and transferred to a nylon membrane. The relative positions of the DNA fragments are preserved during transfer and this membrane can then be used with radioactive probes to determine positions of complementary sequences. Stringency: Degree of mismatch between DNA sequences allowed by reaction conditions, usually governed by temerature of hybridization or salt concentration. Telomere: Physical end of chromosome. Trisomic: Polysomic cell, tissue or individual with one extra chromosome in an otherwise diploid chromosome complement e.g. In man trisomy X (2n = 2x+1 = 46 XXX).

YAC (Yeast Artificial Chromosome): Cloning vector used to clone large fragments of D N A of around 1Mb in a yeast host. The vector, essentially a mini yeast chromosome, carries a multiple cloning site, selectable marker, yeast functional telomeres and small segments which act as a centromere and an origin of replication.

Glossary

References Collins Dictionary of the English Language. ed.: f? Hanks. Pub. Collins, London. Glossary of Genetics and Cytogenetics : Classical and Molecular. 4th edition. Ed. R. Rieger; A. Michaelis; M.M. Green. Pub. SpringerVerlag, Heidelberg. Henderson’s Dictionary of Biological Terms. 9th Edition. Ed. S. Holmes. Pub. Longman, London. Sambrook, J; Fritsch, E.F; Maniatis T (1989) Molecular Cloning : A laboratory Manual. Cold Spring Harbor Laboratory Press.

Glossary

In Situ Hybridization Melody Clark Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Subject Index

Acute myeloid leukaemia, - diagnosis 70-72 - Associated trisomy 7 70 - CD34 antigen 70, 72

Alien chromosome transfer, detection of 4, 5, 8 Alkaline phosphatase/BCIP-NBT 93, 102, 104, 107, 110, 112, 117-119, 157 Alkaline phosphatase/Fast Red 63, 93, 104, 110, 117 Alkaline phosphatase/Vector Red 104, 110, 112 Allopolyploidy 3-5, 8 AMCA 62,78,82,84 Aneuploidy 9,25,67-72,80,84,86 (See also Trisomy) Antibodies Anti-DIG, raised in mouse 63 Anti-DIG conjugated to : alkaline phosphatase 63,93, 102-103, 111, 117, 118 AMCA 62 FlTC 17, 34, 50, 62, 83, 112 horseradish peroxidase 93, 103, 107-108, 110, 117-118 rhodamine 17, 40, 62, 112 Anti-FITC, raised in mouse 62, 83 Anti-FITC conjugated to: alkaline phosphatase 93, 102-103, 107-108, 111, 117 Avididstreptavidin conjugated to: AMCA 62,78,82 CY3 17 FlTC 17, 32-33,38, 50, 61 peroxidase 138 rhodamine 62 Texas red 17,33-34 Biotinylated goat anti-DIG 32 goat anti-avidin 33, 38, 82 Blocking, of 17, 40,47,63, 78-81, 100-102, 106-107, 110, 117-118, CY3 conjugated to: donkey anti-rabbit 80-81 goat anti-mouse 77

rabbit anti-goat 77 rabbit anti-mouse 80-81 - FlTC conjugated to: donkey, anti-mouse 78,83 rabbit anti-mouse 62, 63 swine anti-rabbit 63 - Gold labelled 135-136, 144-145 - Multi-layered system 32-34, 38, 61-63, 69, 78, 82, 135 - Preabsorption 119-120 Antifade mountant 11-13, 17, 33-34,47,61-63,78 Antigen detection 69-72, 80, 86-87 - CD13 antigen 87 - CD19 antigen 80, 82, 86 - CD20antigen 72 - CD3 antigen 71, 72, 77 - CD30 antigen 69-71,86 - CD34antigen 70,72 - CD5 antigen 80, 86 Antisense RNA 91 Autofluorescence 85 Avidin/Streptavidin, conjugated to: - AMCA 62,78,82 - CY3 17 - FITC 1732-33, 38, 50, 61 - peraxidase 138 - rhodamine 62 - Texas red 1733-34 ~~

B-CLL, diagnosis 80 Biotinylated - goat anti-DIG 32 - goat anti-avidin 33, 38, 82 Bleaching, of pigment 116 Blocking - Antibodies 17,40,47, 63, 78-81, 100-102, 106-107, 110, 117-118 - Competitive Hybridization 1, 2, 4, 10, 11, 15, 25, 31, 35 (see also comparative genomic hybridization]

Cot-1 DNA 11,32,33, 37 Human Placental DNA 32,33, 37 CCDcamera 42 CD13 antigen 87 CD19 antigen 80, 82, 86 CD20 antigen 72 CD3 antigen 71, 72, 77 CD30 antigen 69-71, 86 CD34 antigen 70, 72 CD5 antigen 80, 86 Cell culture 31, 35, 59 Cell lines 28 Cell suspension culture 7, 14 Chromosomal in situ suppression 11 Chromosome - Evolution 1,3,5, 7 - Painting 54 - Specific probes 49, 50, 52, 54 - Translocations, detection of 5, 7-8,25,28,34,54, 84 Chronic myeloid leukaemia Colloidal gold 134, 135 Comparative genomic hybridization 1, 6, 11 (See also blocking and competitive hybridization) Competitive hybridization 1, 2, 4, 10, 11, 15, 25, 31, 35 (See also comparative genomic hybridization and blocking) Confocal microscopy 26, 38, 58 Constitutional mosaicism 70 Cosmid - DNA isolation 32 - Labelling 32 - Mapping of 11, 32, 37 - Probe, use as 25 Cot-1 DNA 11, 32,33, 37 Counterstain - DAPl 17,40, 61, 62

M. Clark (Ed.),In Situ Hybridization. 0 Chapman & Hall, Weinheim. ISBN 3-8261-0038-7

-

Propidium iodide 17, 33,38, 58,

61, 62 Rhodamine 17 Texas Red 17 Cryostat sections 68, 75-76,

-

85-86, 112 CTAB 15 CY3, conjugated to : - donkey anti-rabbit 80-81 - goat anti-mouse 77 - rabbit anti-goat 77 - rabbit anti-mouse 80-81 CY3, fluorochrome 17, 78 Cytospin preparations 68-69, 75 DAPl 1740, 61, 62 Dechorionation 98, 114 Denaturation 16,32, 34, 39,49,52,

54, 56, 77, 131-133 Detection systems - Colloidal gold 133-135 - Colourimetric Alkaline phosphatase/ BCIP-NBT 93, 102, 104, 107,

110, 112, 117-119, 157 Alkaline phosphatase/Fast Red 63,93, 104, 110, 117 Alkaline phosphatase/Vector Red 104, 110, 112 Peroxidase/Diaminobenzidine 93, 104, 107, 117-119, 132,

138, 156 - Fluorescent AMCA 78,82,84 CY3 17, 78

ELFTM 93, 104, 112 FlTC 17, 33-34, 38, 61 -63, 78,

84 Rhodamine 40 Texas Red 17,33,34 Dewaxing 116 Digital imaging 7, 74, 83, 88 Digoxygenin - Anti-DIG, raised in mouse 63 - Anti-DIG conjugated to : alkaline phosphatase 63,93,

102-103, 111, 117, 118 AMCA 62 FlTC 17, 34, 50, 62, 83, 112 horseradish peroxidase 93,

103, 107-108, 110, 117-118 rhodamine 1740, 62, 112 Dirvish 28, 29, 38 Dispersed repeats 11 DNA - Dispersed repeats 11 - Genome specific 3 - Repetitive elements 2, 10, 11, 31,

48 Subject Index

- Satellite 3, 10, 50,52, 55, 58 - Species specific 1, 10 DNA halo Preparations 140, 143 DOP-PCR (degenerate oligonucleotide-primed-PCR) 11 Dot d o t - Checking labelling efficiency 155 - Verification of speciesspecificity 10 Double labelling 33-34, 105-111,

117, 134 DPX 95, 105, 119

Gene expression 7, 91, 92 Gene mapping 25,26 Genomesize 2 Genome instability 2, 5, 6 (See also Aneuploidy) Genome specific DNA 3 Genomic DNA - Isolation of DNA 14-15 Glutaraldehyde fixative 92,95,97,99 Glutaraldehyde/Formaldehyde fixative 125, 127-128 Gold grids 125, 129 Gold labelled antibodies 135-136,

144-145 ELFTM 93, 104, 112 EM 6, 144 Embedding - Flat embedding 129 - gelatine capsules 129 - hydrophilic resins Histoclear 105, 114-115 LR White 91, 125, 128-129 paraffin 105, 113, 115-116 tetra hydronapthalene 105 Epifluorescent microscopy 13, 17,

33, 34, 42, 112

Histo-CLEAR 105, 114-116 Hodgkins disease - Aneuploidy 69-71 - CD30 antigen 69-71, 86 - Characterisation of HRS cells 69,

72,86 Human breast carcinoma, detection of 5 Human placental DNA 32, 33, 37 Hybridization mix 16,30, 73-74,

94-95, 100, 124

Erg Oncogene, detection of 28 Evolution 1, 3, 10 Ewings sarcoma, diagnosis of 28

Hybridization chamber 13 Hybrid genome analysis 4,5,6,9

FACS 80 FlTC 17,33-34, 38, 61 -63, 78, 84 - Anti-fluorescein, raised in mouse 62,83 - Anti-fluorescein conjugated to: alkaline phosphatase 93,

lmmunophenotyping (See antigen detection) Imprint preparations 68, 75 Integrated image analysis 144 lnterphase cytogenetic analysis 26-28, 34, 67, 72 lnterspecific hybridization 3, 5, 6 lntrogressive hybridization 5

102-103, 107-108, 111, 117 - conjugated to: donkey, anti-mouse 78,83 rabbit ant-mouse 62, 63 swine anti-rabbit 63 Fixation - formaldehyde 92,97-99 - glutaraldehyde 92,95,97,99 - glutaraldehyde/formaIdehyde 125, 127-128 - paraformaldehyde 12,28,31,59,

77, 82,94, 97-99, 114, 116, 124, 130, 133 Flat embedding 129 Fluorescent microscopy 13, 33,40, 58,78 Formaldehyde 92,97-99 Formvar coated grids 129 Gametogenesis 8 Gelatine capsules 129 Gelatine slide coating 113

Light microscopy 126, 140 LR white embedding 91, 125, 128-129 MAC (morphology, antibody, chromosomes) 68 Malignant B-CELL lymphoma, diagnosis 70 - Associated trisomy X 70 - CD3 antigen 71 Mapping - Cosmids 11, 32, 37 - Genes 25,26 - Yacs 11, 25,27-29 MCAD (medium chain acyl CoA dehydrogenase) 58-59 Metaphase chromosome preparations 14,31, 35,36,56, 59 Microscope slides, coatings - Gelatine 113 - Polylysine 113

- Silane 74-75 - TESPA 113-114 Microscopy - Confocal 26, 38,58 - EM 6, 144 - Epifluorescent 13, 17, 33, 34,42,

112 - Fluorescent 13,33,40,58, 78 - Light 126, 139 - Phase contrast 83 Microslides 56, 58,59, 60, 63 Microtome 113-115 Mountant - antifade 11, 12, 13, 17,33-34,47,

61 -63, 78 - DPX 95, 105, 119 - Temporary, PN buffer 77 mRNA 58 Multiprobe fish 26,33, 53 Multi-layered Antibody system 32-34,38,61-63,69, 78,

82, 135 Myelodysplastic syndrome, diagnosis 71 - Associated Trisomy 8 72

Peroxidase/diaminobenzidine 93,

104, 107, 117-119, 132, 138, 156 Pepsin 124, 130, 131, 133 Phase contrast microscopy 83 Philadelphia chromosome 54, 84 Photography 40,88 Plant breeding 1, 5, 8 Plasmid - Control, use as 15 - Probe, use as 31 Polylysine slide coating 113 Palyploidy 3, 5, 69 Preabsorption, of antibodies 119-120 Primers 48,52, 55,56 Probes - Chromosome specific 49,50,52,

54, 73-74, 84 - Commercial 73-74, 84 - DNA isolation Cosmid 32 Genomic DNA 14-15 YAC 36-37 - Labelling efficiency 155-157 - Labelling Systems Nick translation 14, 126,

147- 149 PCR 49,52,57,59-60,147, Nick translation 14, 126, 147-149 Nuclear architecture 7 Nuclear organisation 1, 7 Oligonucleotide probes 46,48, 49,

133 (see also primers) Osmium tetroxide 128

p53 tumor suppressor gene 6 Pachytene chromosomes 9 Paraffin sections 113, 115 Paraffin embedding 105, 113,

115-116 Paraformaldehydefixative 12,28,

31, 59, 77, 82, 94, 97-99, 114, 116, 124, 130, 133 PCR - DOP-PCR (degenerate oligonucleotide-primed-PCR) 11 - Labelling 49,52,57,59-60, 147,

150-151 - Cycling 50, 52, 57, 60, 151 Permeabilisation - pepsin 124, 130-133 - proteinase K 31, 94-95, 99, 116,

124, 130-131 - saponin 124, 133

150-151 Random Hexamer 32-33,35, 37, 147, 152-153 RNA 96-97 Propidium iodide 17,33,38, 58, 61, 62 Proteinase K 31, 94-95, 99, 116, 124, 130-133 Pulsed field gel electrophoresis 36 RABL 7 Random hexamer 32-33,35,37,

147, 152-153 Recombination - Genomic 5 - lntergenomic 8 Repeat elements 2, 10, 11, 31, 48 - Suppression of 2,4, 10, 11, 15,

- Precautions 96, 133 RNase H 124, 131, 132 Robertsonian translocation 7 Saponin 124, 133 Satellite DNA 3, 10,50,52,55,58 Sectioning 105, 112, 129 Silane slide coating 74-75 Smear preparations 68, 75 Somatic cell hybrids 1,3, 6, 725,

56 Southern blot 2, 9, 11, 15 Spacial organisation 6, 7 Species specific DNA 1, 10 Sporogenesis 8 Statistical analysis 145 Stretched DNA, meoaration 38 Stringency 10, '16, 48, 52, 116,

131-132 Tespa treated slides 113-114 Tetrahydronapthalene 105 Translocations, chromosomal, detection of 5, 7-8,25, 28,34,

54,84 Texas red 17,33,34 Trisomy - Trisomy 7 70 - Trisomy X 70, 71 - Trisomy 8 71, 72 (also see aneuploidy) Tumor analysis 1,3, 6, 67-68 - Southern blot 67, 68 - PCR 67,68 - DNAsequencing 67 - Characterisation of chromosome breakpoints 67 - lnterphase cytogenetics 67 - Chromosome changes 67-70,

72 Ultra thin sections 129, 140

25, 31-35, 37 (see also comparative genomic hybridization, competitive hybridization and blocking) Resolution 26,28 Retrotransposons 3 Reverse chromosome painting 1, 11 Rhodamine 1740 Ribosomal DNA 124, 126 RNA - Labelling 96-97

Wide hybrids, characterisation of 4,5, 8 YAC - DNA isolation 36-37 - Labelling 37 - Physical mapping 11, 25, 27-29 - Probe, use as 25 Yeast cells 36

Subject Index