Fluorescence in situ Hybridization (FISH).

Fluorescence in situ Hybridization (FISH) involves the preparation of two main components: the DNA probe and the target DNA to which the probe will be hybridized. The DNA probe typically comes from cloned sources such as plasmids, cosmids, PACs, YACs, or BACs; where the insert may contain a specific gene or originate from a specific chromosomal locus. Whole-chromosome paints may also be used but are usually applicable to metaphase preparations. The purified DNA can then be labeled and detected indirectly using haptens, or labeled directly using fluorochrome or dye-conjugated nucleotides. Labeling strategies are also variable, employing standard nick translation or PCR labeling methods. The target DNA can take the form of chromosomes spreads or interphase nuclei. The sources of interphase targets may come from cytogenetic preparations or from paraffin-embedded tissues. Both the labeled DNA probe and DNA target are denatured to a single-stranded state and permitted to hybridize to each other. Post-hybridization washes and fluorescently-labeled antibody incubations follow the 24-hour hybridization, and the specimen is ready for visualization by fluorescent microscopy. Successful interpretation of FISH experiments is dependent on the quality of the starting materials, hybridization efficiencies, and stringency of post-hybridization washes and antibody detections.