In situ hybridization is a powerful technology for visualizing the location of specific nucleic acid sequences on chromosomes, single cells, or tissue sections through the use of a nucleic acid probe that is complementary to those sequences and has been labeled in some fashion that renders it detectable. Until the early 1980s, radioisotopes were the only labels available for nucleic acid probes and microautora-diography was the only means to detect in situ-hybridized sequences. Radioactive probes provide limited spatial resolution for in situ hybridization because the decaying particles leave tracks, not discrete spots, in the photographic emulsion. It is further limited by the size of the silver halode crystals in the emulsion. Moreover, many practical inconveniences are imposed by the use of radioactivity, such as the need to observe relatively complicated safety measures, the limited shelf life of radioisotopes, and the long exposure periods required by autoradiography. Finally, with radioactive detection it is not possible to distinguish multiple targets in one multiprobe in situ hybridization experiment.

The development in the 1980s of stable nucleic acid labels that allowed nonradioactive detection through fluorescence or enzyme reactions has demolished these practical and fundamental obstacles. In situ hybridization can now be performed rapidly with multiple differently colored nucleic acid probes at maximum optical resolution, and this has permitted widespread application of this methodology in clinical and basic research. Because of the substantial advantages offered by nonradioactive detection, the presentation of in situ hybridization techniques in this chapter is limited to those methods.

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