Human Sperm Fluorescence In Situ Hybridization (FISH) Protocol

Reference: https://app.jove.com/t/1405/fluoresce...
The protocol for Human Sperm Fluorescence In Situ Hybridization (FISH) is a sophisticated and carefully structured series of steps designed to detect specific DNA sequences within human sperm cells. This advanced technique leverages the use of fluorescently labeled probes that are engineered to bind to complementary DNA sequences, thereby allowing for the visualization of genetic material when observed under a fluorescence microscope.

The process begins with the meticulous preparation of sperm samples, which is a critical step to ensure the integrity and viability of the cells for subsequent analysis. The sperm are typically collected through methods such as masturbation or surgical extraction, and then they are processed to isolate the spermatozoa from seminal fluid. Once isolated, the sperm cells are fixed onto glass slides using a fixative solution, which serves to preserve cellular structure and prevent degradation of the DNA.

Following the fixation step, the slides undergo a denaturation process. This involves heating the slides or treating them with a chemical solution to separate the double-stranded DNA into single strands. This denaturation is essential as it creates accessible sites for the fluorescent probes to bind to their complementary sequences.

After denaturation, the slides are incubated with the fluorescent probes, which are specifically designed to target particular regions of interest within the sperm DNA. These probes are labeled with fluorescent dyes that emit light of specific wavelengths when excited by the appropriate light source. The hybridization step allows the probes to bind to their complementary DNA sequences, forming stable probe-DNA complexes.