Fluorescence In Situ Hybridization (FISH) is a technique used to visualize specific DNA or RNA sequences within cells or tissues. FISH method utilized to diagnose genetic diseases, gene mapping, and identification of chromosomal abnormalities, and may also be used to study comparisons among the chromosomes' arrangements of genes of related species.
1) DNA or RNA probe specific to the target sequence
2) Fixative (e.g., paraformaldehyde or methanol)
3) Permeabilization solution (e.g., 0.1% Triton X-100 in PBS)
4) Denaturation solution (e.g., 70% formamide in 2X SSC)
5) Hybridization buffer (e.g., 50% formamide in 2X SSC)
6) Mounting medium with DAPI or another nuclear stain
7) Microscope slides
1) Preparation of cells or tissue sections: Grow cells on microscope slides or mount tissue sections onto microscope slides.
2) Fix cells or tissues with a fixative, such as paraformaldehyde or methanol, for 10-20 minutes at room temperature.
3) Rinse cells or tissue sections with PBS.
4) Permeabilization: Incubate cells or tissue sections with permeabilization solution to allow the probe to enter the cells. Incubate for 10-20 minutes at room temperature.
5) Denaturation: Incubate cells or tissue sections with denaturation solution to separate the double-stranded DNA or RNA into single strands. Incubate for 10-20 minutes at 75-80°C.
6) Hybridization: Dilute the DNA or RNA probe in hybridization buffer to a concentration appropriate for your specific probe and incubate cells or tissue sections with the probe solution overnight at 37°C.
7) Post-hybridization wash: Wash cells or tissue sections with a series of wash solutions to remove unbound probe and reduce background staining.
8) Nuclear staining: If using DAPI or another nuclear stain, add the staining solution to the cells or tissue sections for 10-15 minutes.
9) Mounting: Mount the slides using mounting medium with a coverslip and seal with nail polish.
11) Observe and capture images using a fluorescence microscope.
It is important to include appropriate controls, such as a negative control without probe or a probe against a different target sequence, to ensure the specificity of the staining. The specific details of the FISH protocol may vary depending on the type of cells or tissue and the specific probe used.