Fluorescence in situ hybridization
In situ hybridization (ISH) is used to visualize defined nucleic acid sequences in cellular preparations by hybridization of complementary probe sequences. Probe sequences can be labeled with isotopes, but nonisotopic ISH is used increasingly as it is considerably faster, usually has greater signal resolution, and provides many options to simultaneously visualize different targets by combining various detection methods. The most popular protocols use fluorescence detection, as described here. These protocols have many applications, from basic gene mapping and diagnosis of chromosomal aberrations, to detailed studies of cellular structure and function, such as the painting of chromosomes in three-dimensionally preserved nuclei. This protocol describes fluorescence in situ hybridization (FISH) of biotin- or digoxigenin-labeled probes to denatured metaphase chromosomes and interphase nuclei. The hybridized probes are detected and visualized using fluorochrome-conjugated reagents.
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