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Nonradioactive In Situ Hybridization
Application Manual




To select a chapter section, click on the corresponding page number.
The chapters below are in Acrobat PDF format .

Chapter 1
General Introduction to In Situ Hybridization General Introduction to In Situ Hybridization 9
Introduction to Hapten Labeling and Detection of Nucleic Acids 11
Choosing the Right Labeling Method for your Hybridization Experiment 15

Chapter 2
Guidelines for In Situ Hybridization Guidelines for In Situ Hybridization 21
Details of the Technique 22

Chapter 3
Nucleic Acid Hybridization - General Aspects Nucleic Acid Hybridization - General Aspects 33

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 41
Random primed labeling of ds DNA with DIG-, Biotin- or Fluorescein-High Prime reaction mix 41
PCR labeling of ds DNA with the PCR DIG Probe Synthesis Kit or PCR Labeling Mixes 43
Nick-translation labeling of ds DNA with Nick Translation Mixes for in situ Probes 49
Nick-translation labeling of ds DNA with DIG-, Biotin-, or Fluorochrome-labeled dUTP 52
RNA labeling by in vitro transcription of DNA with DIG, Biotin or Fluorescein RNA Labeling Mix 54
Oligonucleotide 3'-end labeling with DIG-ddUTP, Biotin-ddUTP, or Fluorescein-ddUTP 58
Oligonucleotide tailing with a DIG-dUTP, Biotin-dUTP, or Fluorescein-dUTP mixture 60
Chem-Link labeling of DNA or RNA with DIG or Biotin 63
Estimating the Yield of DIG-labeled Nucleic Acids 65
Purification of labeled probes using the High Pure PCR Product Purification Kit 71

Chapter 5
Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections Procedures for In Situ Hybridization to whole chromosomes 77
In situ hybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates 78
Detection of chromosomal imbalances using DOP-PCR and comparative genomic hybridization (CGH) 95
Fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension 104
A simplified and efficient protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG-labeled DNA probe 108
Multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems 112
DNA in situ hybridization with an alkaline phosphatase-based fluorescent detection system 124
Combined DNA in in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations 127
In situ hybridization to mRNA in in vitro cultured cells with DNA probes 135
Identification of single bacterial cells using DIG-labeled oligonucleotides 139
Detection of HPV11 DNA in paraffin-embedded laryngeal tissue with a DIG-labeled DNA probe 143
Detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probes 149
Detection of mRNA on paraffin-embedded material of the central nervous system with DIG-labeled RNA probes 164
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 172
Detection of neuropeptide mRNAs in tissue sections using oligonucleotides tailed with fluorescein-12-dUTP or DIG-dUTP 178
RNA in situ hybridization using DIG-labeled cRNA probes 181
Detection of mRNAs on cryosections of the cardiovascular system using DIG-labeled RNA probes 189
Molecular and Biochemical Analysis of Arabidopsis 197
Whole mount in situ hybridization for the detection of mRNA in Drosophila embryos 208
Detection of even-skipped transcripts in Drosophila embryos with PCR/DIG-labeled DNA probe 216
Whole mount fluorescence in situ hybridization (FISH) of repetitive DNA sequences on interphase nuclei of the small cruciferous plant Arabidopsis thaliana 220

Chapter 6
Ordering Information 231

Chapter 7
Index 237