Chromogenic In Situ Hybridization in Tumor Pathology

Ever since the correlation was found between the pathogenesis of diseases and genomic alterations, molecular cytogenetic techniques have found a place in molecular medicine. These techniques are used in tracing gene and genomic abnormalities that are underlying in the development of cancer and genetic diseases. In 1969, Gall and Pardue introduced a technique known as “in situ hybridization” (ISH) to localize nucleic acids in individual cells (1 ). At that time, the capabilities of ISH were limited to highly repetitive sequences using radioactively labeled probes that were subsequences visualized by autoradiography. The use of radioisotopes has many disadvantages and has been replaced in DNA ISH by nonradioactive detection methods. The most commonly used reporter molecules are haptens, such as biotin and digoxigenin, which can be incorporated easily in the probe DNA. The tagged probes are then detected with labeled antibodies against the specific tag or, as in the case of biotin, with a labeled avidin molecule. Since the first report of fluorescent in situ hybridization (FISH) (2 ), the principle of FISH has remained essentially the same, with the exception that biotin and digoxigenin have partly been replaced by directly fluorochrome-conjugated nucleotides, which simplifies the laboratory protocol.