In Situ Hybridization Protocols

The technique of in situ hybridization, in its various forms, has been used routinely in many laboratories for a number of years. In the post-genome era, gene arrays and proteomics have allowed us to identify hitherto unknown unrecognized pathways and mechanisms. However, rather than diminish the importance of in situ hybridization, the now widespread use of screening te- nologies has increased the need to temporally and spatially localize the dist- bution of mRNA expression. Our intention, in In Situ Hybridization Protocols is to provide ample inf- mation for novices planning to set up the in situ hybridization technique and use it in their laboratory for the first time, as well as giving updates of recent developments for those laboratories where in situ hybridization techniques are already in use. Despite its widespread significance, in situ hybridization has retained a re- tation as one of the more difficult and capricious molecular biological te- niques. This may in part be because of the hybrid nature of the technique, which often requires a mixture of molecular biological and histological skills. The two techniques are usually taught and acquired in different streams of biolo- cal science. The step-by-step and detailed protocols provided in In Situ Hybridization Protocols by researchers active in the field should make it p- sible for both the molecular biologist with little experience of histology and the histologist with little experience of molecular biology to use the technique s- cessfully in their laboratories.

In In Situ Hybridization, Third Edition, Ian Darby updates his highly successful second edition and shifts the focus to tissue and cell in situ hybridization. Drawing on experts working in diverse areas and taking advantage of advances in non-isotopic in situ hybridization and amplification techniques, the authors describe in detail their readily reproducible techniques for preparation, detection, and tissue hybridization. Among the new techniques detailed are PNA probes for viral diagnostics, plant in situ hybridization, cell proliferation detection, and quantitation of in situ hybridization. There are also cutting-edge techniques for tissue microarrays, expanded embryology-developmental gene detection, and expanded cell culture. Derivative techniques presented include the identification of transplanted cells, histones, nick-end labeling for apoptosis, the use of peptide nucleic acid probes, and in situ hybridization of plant specimens. The protocols follow the successful Methods in Molecular Biology series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Completely updated and highly practical, In Situ Hybridization, Third Edition allows both the molecular biologist with little experience of histology and the histologist with little experience of molecular biology to use these powerful new techniques successfully.

Treatment of Tissue Sections for In Situ Hybridization.- Preparation of Template DNA and Labeling Techniques.- In Situ Hybridization Using cRNA Probes.- Tyramide Signal Amplification for DNA and mRNA In Situ Hybridization.- Expression Analysis of Murine Genes Using In Situ Hybridization With Radioactive and Nonradioactively Labeled RNA Probes.- Nonradioactive In Situ Hybridization on Frozen Sections and Whole Mounts.- In Situ Hybridization of Whole-Mount Embryos.- In Situ Detection of Epstein-Barr Virus and Phenotype Determination of EBV-Infected Cells.- PNA-In Situ Hybridization Method for Detection of HIV-1 DNA in Virus-Infected Cells and Subsequent Detection of Cellular and Viral Proteins.- Electron Microscopy and In Situ Hybridization.- In Situ Hybridization Using Riboprobes on Free-Floating Brain Sections.- An In Situ Hybridization Technique to Detect Low-Abundance Slug mRNA in Adherent Cultured Cells.- Identification of Transplanted Human Cells in Animal Tissues.- In Situ Hybridization to Plant Tissues and Chromosomes.- Histochemical Localization of Cell Proliferation Using In Situ Hybridization for Histone mRNA.- Histochemical Localization of Apoptosis With In Situ Labeling of Fragmented DNA.- The Use of Combined Immunohistochemical Labeling and In Situ Hybridization to Colocalize mRNA and Protein in Tissue Sections.- Semiquantitative In Situ Hybridization Using Radioactive Probes to Study Gene Expression in Motoneuron Populations.- Quantitative In Situ Hybridization of Tissue Microarrays.

New Third Edition Focuses
on Tissue In Situ Hybridization