Nonradioactive In Situ Hybridization Appl. Manual
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Chapter 1
General Introduction to In Situ Hybridization 		General Introduction to In Situ Hybridization 8
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 18
Details of the Technique 18


Chapter 3
Nucleic Acid Hybridization - General Aspects 		Nucleic Acid Hybridization - General Aspects 26


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 36
Random primed labeling of ds DNA with DIG-, Biotin- or Fluorescein-High Prime reaction mix 36
PCR labeling of ds DNA with the PCR DIG Probe Synthesis Kit or PCR Labeling Mixes 38
Nick-translation labeling of ds DNA with Nick Translation Mixes for in situ Probes 45
Nick-translation labeling of ds DNA with DIG-, Biotin-, or Fluorochrome-labeled dUTP 47
RNA labeling by in vitro transcription of DNA with DIG, Biotin or Fluorescein RNA Labeling Mix 49
Oligonucleotide 3'-end labeling with DIG-ddUTP, Biotin-ddUTP, or Fluorescein-ddUTP 53
Oligonucleotide tailing with a DIG-dUTP, Biotin-dUTP, or Fluorescein-dUTP mixture 56
Estimating the Yield of DIG-labeled Nucleic Acids 59
Purification of labeled probes using the High Pure PCR Product Purification Kit 65


Chapter 5
Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections 		Procedures for In Situ Hybridization to whole chromosomes 66
In situ hybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates 69
Fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension 85
A simplified and efficient protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG-labeled DNA probe 89
Multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems 94
DNA in situ hybridization with an alkaline phosphatase-based fluorescent detection system 105
Combined DNA in in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations 108
In situ hybridization to mRNA in in vitro cultured cells with DNA probes 116
Identification of single bacterial cells using DIG-labeled oligonucleotides 119
Detection of HPV11 DNA in paraffin-embedded laryngeal tissue with a DIG-labeled DNA probe 123
Detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probes 129
Detection of mRNA on paraffin-embedded material of the central nervous system with DIG-labeled RNA probes 144
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 151
Detection of neuropeptide mRNAs in tissue sections using oligonucleotides tailed with fluorescein-12-dUTP or DIG-dUTP 157
RNA in situ hybridization using DIG-labeled cRNA probes 160
Detection of mRNAs on cryosections of the cardiovascular system using DIG-labeled RNA probes 166
Molecular and Biochemical Analysis of Arabidopsis 176
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 193
Whole mount fluorescence in situ hybridization (FISH) of repetitive DNA sequences on interphase nuclei of the small cruciferous plant Arabidopsis thaliana 197


Chapter 6
Ordering Information 208


Chapter 7
Index 220