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Tables of Contents for In Situ Hybridization
Chapter/Section Title
Page #
Page Count
List of contributors
xv
Abbreviations
xvii
The theory and practice of in situ hybridization
1
22
David G. Wilkinson
Introduction
1
4
Preparation of Probe
5
5
Type of probe and method of synthesis
7
1
Length of probe
8
1
Label
9
1
Preparation of tissue
10
1
Fixation
10
1
Embedding and sectioning of tissue
10
1
Subbing of Slides
11
1
Pre-treatments of tissue
11
1
Hybridization and washing
12
2
Optimal conditions for hybridization
12
2
Post-hybridization washing
14
1
Visualization of Signal
14
4
Radioactive probes
14
1
Hapten labelled probes
15
1
Double staining techniques
16
2
Controls and troubleshooting
18
2
Photography
20
3
Acknowledgements
21
1
References
21
2
Oligonucleotide probes for in situ hybridization
23
46
Marcus Rattray
Gregory J. Michael
Introduction
23
1
Design of oligonucleotide probes
23
6
Theoretical considerations
23
4
Design in practice
27
2
Oligonucleotide probe synthesis
29
4
Manufacturing oligonucleotides
29
1
Oligonucleotide purification
29
3
Quantification of oligonucleotide
32
1
Oligonucleotide labelling
33
6
Choice of labelling method
33
1
3' end-labelling with radiolabelled nucleotides
34
4
Non-radioactive 3' end-labelling
38
1
In situ hybridization with oligonucleotides
39
16
Tissue preparation
39
6
Hybridization and post-hybridization washes
45
6
Combined in situ hybridization with immunocytochemistry
51
2
Controls for in situ hybridization
53
2
Detection of hybrids and quantification
55
9
Detection of radiolabelled probes
55
6
Imaging and analysis of sections hybridized with radiolabelled probes
61
3
Image analysis using non-radioactive probes
64
1
Stock solutions and precautions against RNase contamination
64
5
References
66
3
Detection of mRNA in tissue sections with radiolabelled riboprobes
69
18
Antonio Simeone
Introduction
69
1
Recovery and fixation of embryos
69
2
Wax embedding, orientation, and sectioning
71
4
Probe preparation
75
1
Pre-treatment and hybridization
76
2
Post-hybridization washes
78
1
Autoradiography
79
1
Developing, staining, and photography
80
2
In situ hybridization on cryosections
82
1
Useful molecular markers to define gene expression patterns
83
4
Acknowledgements
84
1
References
84
3
In situ hybridization of mRNA with hapten labelled probes
87
20
Qiling Xu
David G. Wilkinson
Introduction
87
1
When to hybridize to sections or whole mounts
87
1
General strategy
88
1
Whole mount in situ hybridization
89
10
Preparation of labelled RNA probe
90
2
Fixation and pre-treatment of embryos
92
3
Hybridization, washing, and detection of probe
95
4
In situ hybridization to tissue sections or cultured cells
99
5
General precautions
99
1
Preparation of tissue sections or cultured cells on slides
100
1
Pre-treatments, hybridization, washing, and detection of probe
101
3
Photography
104
3
Acknowledgements
106
1
References
106
1
Two colour in situ hybridization
107
20
T. Jowett
Methods of localization of multiple transcripts
107
14
Two colour in situ hybridization with chromogenic substrates for alkaline phosphatese and horse-radish peroxidase
110
3
Two colour in situ hybridization with chromogenic substrates for alkaline phosphatase
113
4
Double fluorescent in situ hybridization
117
4
Methods for simultaneous in situ localization of transcripts and tissue antigens
121
6
Indirect immunolocalization of a tissue antigen
122
1
Immunolocalization by the peroxidase anti-peroxidase (PAP) method
123
1
Immunolocalization by the ABC method
124
2
References
126
1
Electron microscope in situ hybridization: the non-isotopic post-embedding procedure
127
34
Francine Puvion-Dutilleul
Introduction
127
1
Preparation of biological material for ultrathin sectioning
128
3
Fixation
128
1
Dehydration and Lowicryl embedding
129
1
Supports for ultrathin sections
130
1
Sectioning and recovering ultrathin sections
131
1
Probes
131
2
Generalities
131
1
Probe choice
131
2
Hybridization
133
5
Generalities
133
1
Hybridization solution
133
1
Heat treatment of the hybridization solutions
134
1
Formation of hybrids on ultrathin sections
134
2
Post-hybridization steps
136
1
Hybridization protocol
137
1
Specific preparations of target molecules prior to hybridization
138
3
Why and how does one improve the accessibility of the probe to the target?
138
2
Why and how to perform nuclease digestion(s) prior to hybridization
140
1
Controls of the specificity of the detection
141
1
Routine protocols for specific detection of defined nucleic a cid sequences and applications
142
13
Specific detection of DNA sequences by using biotinylated double-stranded DNA probes
142
6
Specific detection of RNA sequences by using biotinylated probes
148
7
Double detections
155
1
Simultaneous detection of two distict nucleic acid sequences
155
1
Simultaneous detection of a nucleic acid sequence and an antigen
156
1
Conclusions
156
5
Acknowledgements
157
1
References
157
4
Detection of genomic sequences by fluorescence in situ hybridization to chromosomes
161
29
Lyndal Kearney
Introduction
161
1
FISH: practical considerations
162
14
Preparation of metaphase chromosomes
162
1
Probes for FISH
163
4
Pre-treatment of chromosomal DNA
167
1
Competitive in situ suppression hybridization
168
3
Detection of bound, labelled probe
171
3
Visualization of signal
174
2
Troubleshooting
176
1
Applications
177
13
Gene mapping
177
2
Identification of genetic material of unknown origin
179
3
Comparative genomic hybridization (CGH)
182
1
Breakpoint identification
183
1
Interphase analysis
184
1
Comparative mapping
184
1
Gene expression, structure, and function
185
5
References
190
1
Gene expression databases
190
25
Duncan Davidson
richard Baldock
Jonathan Bard
Mathew Kaufman
Joel E. Richardson
Janan T. Eppig
Martin Ringwald
Introduction
190
1
The database approach: combining information in a common format
190
3
Descriptions of embryos: information formats for developmental biology
190
1
Molecular anatomy: a new description of the embryo
191
1
Using gene expression data to build molecular pathways
191
1
Linking pathways with processes at the cell and tissue levels
192
1
Problems with the database approach
192
1
Outline survey of gene expression databases
193
12
Caenorhabditis
194
1
Drosophila
194
1
Zebrafish
195
1
Xenopus
196
1
Mouse
196
8
Human
204
1
Specialist data
205
1
Using gene expression databases
205
7
Practical considerations in preparing to enter data into a database
205
6
Querying the database
211
1
The future of gene expression databases
212
3
Between-species links
213
1
Acknowledgements
213
1
References
214
1
Appendix
215
6
Index
221