In Situ Hybridization to Human Chromosomes of an Alkaline Phosphatase-Labeled Centromeric Probe

The application of in situ hybridization and the underlying rationale are described in Chapter 46. In situ hybridization of nucleic acid probes to cellular targets of biological specimens affixed to a slide has benefited greatly from the concepts and strategies so elegantly developed under the umbrella of immunocytochemistry. These include direct and indirect affinity assays involving biotinylated nucleic acid probes and streptavidin complexed to an enzyme, indirect immunoassays and indirect immunoaffinity assays involving antibodies that recognize an antigen conjugated to a nucleic acid probe, and direct assays in which enzyme- or fluor-labeled probes are used (1 ). The selection of a specific probe-detection system is usually dictated by the required sensitivity and resolution. For example, the indirect immunoassay and the indirect immunoaffinity assay use primary and secondary antibodies, thereby facilitating the formation of a branched network of antibodies and an increase in the amount of label that indirectly becomes associated with the probe-target hybrid. Fluorescent tags provide the greatest resolution, thereby optimizing localization. By using two or more fluorochromes simultaneously for two or more cellular targets, it is possible to discern the precise location of multiple targets in the same specimen (2 ).