in situ Hybridization Protocols for Detection of Viral DNA Using Radioactive and Nonradioactive DNA Probes

in situ hybridization can provide accurate intracellular localization of specific viral nucleic acids in infected tissues and cells. The technique, although conceptually simple, is affected by many variables including: stability and accessibility of target sequences; methods of tissue and cell fixation; prehybridization and hybridization conditions; choice of indicator molecules and detection system; and size, specific activity, and nature of the probe used. Each new tissue and target sequence may have different optimal conditions for each of these variables and should be carefully balanced to yield the maximum amount of information. The technique has been used extensively in studies of viral infection of cells. Particular advantages include: (1) high sensitivity when examining tissues where a small percentage of cells contain a high copy of target molecules; (2) ability to correlate target sequences with cell type and distribution, histological features, and intracellular localization (nuclear vs cytoplasmic).