In Situ Hybridization

Transcription

1 In Situ Hybridization A Practical Approach Second Edition Edited by D. G. WILKINSON MMfl, London Oxford New York Tokyo OXFORD UNIVERSITY PRESS 1998

2 List of con trib u tors Abbreviations 1. The theory and practice of in situ hybridization 1 David G. Wilkinson t_ 1. Introduction 1 2. Preparation of probe 5 Type of probe and method of synthesis 7 Length of probe 8 Label 9 3. Preparation of tissue 10 Fixation 10 Embedding and sectioning of tissue 10 Subbing of slides Pre-treatments of tissue Hybridization and washing 12 Optimal conditions for hybridization 12 Post-hybridization washing Visualization of signal 14 Radioactive probes 14 Hapten labelled probes 15 Double staining techniques Controls and troubleshooting Photography 20 Acknowledgements 21 References Oligonucleotide probes for in situ hybridization 23 Marcus Rattray and Gregory J. Michael 1. Introduction 23

3 2. Design of oligonucleotide probes 23 Theoretical considerations 23 Design in practice Oligonucleotide probe synthesis 29 Manufacturing oligonucleotides 29 Oligonucleotide purification 29 Quantification of oligonucleotide Oligonucleotide labelling 33 Choice of labelling method 33 3' end-labelling with radiolabelled nucleotides 34 Non-radioactive 3' end-labelling In situ hybridization with oligonucleotides 39 Tissue preparation 39 Hybridization and post-hybridization washes 45 Combined in situ hybridization with immunocytochemistry 51 Controls for in situ hybridization Detection of hybrids and quantification 55 Detection of radiolabelled probes '55 Imaging and analysis of sections hybridized with radiolabelled probes 61 Image analysis using non-radioactive probes Stock solutions and precautions against RNase contamination 64 References Detection of mrna in tissue sections with radiolabelled riboprobes 69 Antonio Simeone 1. Introduction Recovery and fixation of embryos Wax embedding, orientation, and sectioning Probe preparation Pre-treatment and hybridization Post-hybridization washes Autoradiography Developing, staining, and photography In situ hybridization on cryosections Useful molecular markers to define gene expression patterns 83 Acknowledgements 84 References 84

4 4. In situ hybridization of mrna with hapten labelled probes 87 Qiling Xu and David G. Wilkinson 1. Introduction When to hybridize to sections or whole mounts General strategy Whole mount in situ hybridization 89 Preparation of labelled RNA probe 90 Fixation and pre-treatment of embryos 92 Hybridization, washing, and detection of probe In situ hybridization to tissue sections or cultured cells 99 General precautions 99 Preparation of tissue sections or cultured cells on slides 100 Pre-treatmentS) hybridization, washing, and detection of probe Photography 104 Acknowledgements 106 References Two colour in situ hybridization 107 T. Jowett 1. Methods of localization of multiple transcripts 107 Two colour in situ hybridization with chromogenic substrates for alkaline phosphatase and horse-radish peroxidase 110 Two colour in situ hybridization with chromogenic substrates for alkaline phosphatase 113 Double fluorescent in situ hybridization Methods for simultaneous in situ localization of transcripts and tissue antigens 121 Indirect immunolocalization of a tissue antigen 122 Immunolocalization by the peroxidase anti-peroxidase (PAP) method 123 Immunolocalization by the ABC method 124 References ^ Electron microscope in situ hybridization: the non-isotopic post-embedding procedure 127 Francine Puvion-Dutilleul 1. Introduction 127 xi

5 2. Preparation of biological material for ultrathin sectioning 128 Fixation 128 Dehydration and Lowicryl embedding 129 Supports for ultrathin sections 130 Sectioning and recovering ultrathin sections Probes 131 Generalities 131 Probe choice Hybridization 133 Generalities 133 Hybridization solution 133 Heat treatment of the hybridization solutions 134 Formation of hybrids on ultrathin sections 134 Post-hybridization steps 136 Hybridization protocol Specific preparations of target molecules prior to hybridization 138 Why and how does one improve the accessibility of the probe to the target? 138 Why and how to perform nuclease digestion(s) prior to hybridization Controls of the specificity of the detection Routine protocols for specific detection of defined nucleic acid sequences and applications 142 Specific detection of DNA sequences by using biotinylated doublestranded DNA probes 142 Specific detection of RNA sequences by using biotinylated probes Double detections 155 Simultaneous detection of two distinct nucleic acid sequences 155 Simultaneous detection of a nucleic acid sequence and an antigen Conclusions 156 Acknowledgements 157 References Detection of genomic sequences by fluorescence in situ hybridization to chromosomes i6i Lyndal Kearney 1. Introduction FISH: practical considerations 162 Preparation of metaphase chromosomes 162 xii

6 Probes for FISH 163 Pre-treatment of chromosomal DNA 167 Competitive in situ suppression hybridization 168 Detection of bound, labelled probe 171 Visualization of signal Troubleshooting Applications 177 Gene mapping 177 Identification of genetic material of unknown origin 179 Comparative genomic hybridization (CGH) 182 Breakpoint identification 183 Interphase analysis 184 Comparative mapping 184 Gene expression, structure, and function 185 References Gene expression databases 190 Duncan Davidson, Richard Baldock, Jonathan Bard, Matthew Kaufman, Joel E. Richardson, Janan T. Eppig, and Martin Ringwald 1. Introduction The database approach: combining information in a common format 190 Descriptions of embryos: information formats for developmental biology 190 Molecular anatomy: a new description of the embryo 191 Using gene expression data to build molecular pathways 191 Linking pathways with processes at the cell and tissue levels 192 Problems with the database approach Outline survey of gene expression databases 193 Caenorhabditis 194 Drosophila 194 Zebrafish tf 195 Xenopus 196 Mouse 196 Human 204 Specialist data Using gene expression databases 205 Practical considerations in preparing to enter data into a database 205 Querying the database The future of gene expression databases 212 Between-species links 213 xiii

7 Acknowledgements 213 References 214 Appendix 215 Index 221 XIV