Denaturation and Hybridization
Double-strand DNA denaturation of the sperm and FISH probes is carried out after incubation at high temperature (70°C-74°C). After denaturation, both DNAs are coincubated and hybridized to form a duplex of complementary strands. Hybridization protocols vary according to the type of FISH probe used, requiring different times and temperatures of hybridization (commonly between 4 and 16 hours at 37°C-42°C).
FISH analysis on sperm is commonly performed using centromeric, locus-specific, and subtelomeric fluorescent DNA probes. For segregation studies in structural rearrangements, specific combinations of these three types of probes are designed for each specific rearrangement. However, in carriers of numerical sex chromosome abnormalities and also in normal karyotype infertile men, the most widely analyzed are chromosomes 13, 18, 21, X, and Y using centromeric and locus-specific probes.
Excess DNA probes hybridized to unspecific complementary sequences are removed by astringent washes at high temperatures and low saline concentrations (e.g., with saline sodium citrate [SSC]). Finally, a counterstain is applied to allow the visualization of the sperm nucleus (e.g., 4',6-diamino-2-fenilindol [DAPI] or DAPI/Antifade).