Nonradioactive In Situ Hybridization
Application Manual
Last Update April 26, 2000
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Chapter 1
General Introduction
to In Situ
Hybridization
General Introduction to In Situ Hybridization
7
Introduction to Hapten Labeling and Detection of
Nucleic Acids
10
Choosing the Right Labeling Method for your
Hybridization Experiment
13
Chapter 2
Guidelines for In Situ
Hybridization
Guidelines for In Situ Hybridization
17
Details of the Technique
19
Chapter 3
Nucleic Acid
Hybridization General Aspects
Nucleic Acid Hybridization - General Aspects
27
Chapter 4
Procedures for
Labeling DNA, RNA,
and Oligonucleotides
with DIG, Biotin, or
Fluorochromes
Procedures for Labeling DNA, RNA, and
Oligonucleotides with DIG, Biotin, or
Fluorochromes
33
Random primed labeling of ds DNA with DIG-, Biotinor Fluorescein-High Prime reaction mix
33
PCR labeling of ds DNA with the PCR DIG Probe
Synthesis Kit or PCR Labeling Mixes
36
Nick-translation labeling of ds DNA with Nick
Translation Mixes for in situ Probes
40
Nick-translation labeling of ds DNA with DIG-,
Biotin-, or Fluorochrome-labeled dUTP
42
RNA labeling by in vitro transcription of DNA with
DIG, Biotin or Fluorescein RNA Labeling Mix
44
Oligonucleotide 3'-end labeling with DIG-ddUTP,
Biotin-ddUTP, or Fluorescein-ddUTP
46
Oligonucleotide tailing with a DIG-dUTP,
Biotin-dUTP, or Fluorescein-dUTP mixture
47
Purification of labeled probes using the High Pure
PCR Product Purification Kit
50
Estimating the Yield of DIG-labeled Nucleic Acids
51
Chapter 5
Procedures for In Situ
Hybridization to
Chromosomes, Cells,
and Tissue Sections
Procedures for In Situ Hybridization to whole
chromosomes
57
In situ hybridization to human metaphase
chromosomes using DIG-, biotin-, or
fluorochrome-labeled DNA probes and detection with
fluorochrome conjugates
62
Detection of chromosomal imbalances using
DOP-PCR and comparative genomic hybridization
(CGH)
72
Identification of chromosomes in metaphase spreads
and interphase nuclei with PRINS Oligonucleotide
Primers and DIG- or rhodamine-labeled nucleotides
79
Identification of chromosomes in metaphase spreads
with DIG- or fluorescein-labeled human
chromosome-specific satellite DNA probes
88
Fluorescence in situ hybridization of a repetitive
DNA probe to human chromosomes in suspension
94
A simplified and efficient protocol for nonradioactive
in situ hybridization to polytene chromosomes with a
DIG-labeled DNA probe
97
Multiple-target DNA in situ hybridization with
enzyme-based cytochemical detection systems
100
DNA in situ hybridization with an alkaline
phosphatase-based fluorescent detection system
108
Procedures for In Situ Hybridization to cells
110
Combined DNA in in situ hybridization and
immunocytochemistry for the simultaneous
detection of nucleic acid sequences, proteins, and
incorporated BrdU in cell preparations
110
In situ hybridization to mRNA in in vitro cultured
cells with DNA probes
116
Identification of single bacterial cells using
DIG-labeled oligonucleotides
119
Procedures for In Situ Hybridization to tissues
122
Detection of HPV11 DNA in paraffin-embedded
laryngeal tissue with a DIG-labeled DNA probe
122
Detection of mRNA in tissue sections using
DIG-labeled RNA and oligonucleotide probes
126
Detection of mRNA on paraffin embedded material of
the central nervous system with DIG-labeled RNA
probes
136
RNA-RNA in situ hybridization using DIG-labeled
probes: the effect of high molecular weight polyvinyl
alcohol on the alkaline phosphatase indoxyl-nitroblue
tetrazolium reaction
141
Detection of neuropeptide mRNAs in tissue sections
using oligonucleotides tailed with
fluorescein-12-dUTP or DIG-dUTP
146
In situ hybridization of DIG-labeled rRNA probes to
mouse liver ultrathin sections
148
RNA in situ hybridization using DIG-labeled cRNA
probes
152
Procedures for In Situ Hybridization to Whole
mount ISH
158
Localization of the expression of the segmentation
gene hunchback in Drosophila embryos with
DIG-labeled DNA probes
158
Detection of even-skipped transcripts in Drosophila
embryos with PCR/DIG-labeled DNA probes
162
Whole mount fluorescence in situ hybridization
(FISH) of repetitive DNA sequences on interphase
nuclei of the small cruciferous plant Arabidopsis
thaliana
165
Chapter 6
References
173
Chapter 7
Ordering Information
201
Chapter 8
Index
207
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