Fluorescence In Situ Hybridization (FISH) Protocol

• Fluorescence in situ hybridization (FISH) is an emerging molecular cytogenetics technique, which is a non-radioactive in situ hybridization technique developed in the late 1980s based on the original radioactive in situ hybridization technique. It has been widely used in many fields such as genome structure study of plants and animals, chromosome fine structure variation analysis, virus infection analysis, human prenatal diagnosis, tumor genetics, and genome evolution study.
• The basic principle of FISH is to use a known labeled single-stranded nucleic acid as a probe to specifically bind to an unknown single-stranded nucleic acid in the material to be examined according to the principle of base complementarity to form a hybridized double-stranded nucleic acid that can be detected. Since DNA molecules are arranged linearly along the longitudinal axis of chromosomes, the probes can be hybridized directly into chromosomes to localize specific genes on the chromosomes.

Slide Preparation

• Start with chromosome preparations from any cell type.
• Incubate with 200 µL RNase for 1 hour at 37°C
• Wash slides in 2xSSC for 5 minutes. Repeat.
• Rinse slides in 10 mM HCl.
• Incubate with 200 µL pepsin for 10 minutes at 37°C.
• Rinse slides in deionized H₂O.
• Wash slides in 2xSSC for 5 minutes. Repeat.
• Stabilize slides in paraformaldehyde for 10 minutes.
• Wash slides in 2x SSC for 5 minutes. Repeat.
• Dehydrate slides in an ethanol series, 70%, 80%, 95%, 2 minutes each.
• Air dry.

Hybridization

• Prepare 30 µl hybridization solution per slide. Heat to 70°C. for 10 minutes and place on ice.
• Place 30 µl of hybridization solution on each slide and cover it with a plastic cover slip.
• Denature slide at 65-70°C for 5 minutes on heat block.
• Gradually decrease the temperature to 37°C.
• Hybridize at 37°C overnight in a humidity chamber.

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Detection

• Wash slides in 2xSSC to remove coverslip.
• Wash slides in wash buffer at 40°C for 5 minutes. Repeat.
• Wash slides in 0.1xSSC at 40°C for 5-15 minutes.
• Wash slides in 2xSSC at 40°C for 5-15 minutes.
• Cool slides to room temperature.
• Equilibrate slides in the detection buffer for 5 minutes.
• Block in blocking buffer for 20-30 minutes.
• Incubate with 50 µl antibody or detection compound for 30-60 minutes (e.g., 5 µg/ml Streptavidin-Cy3 in blocking buffer).
• Wash slides in 2xSSC for 5 minutes. Repeat twice.
• Counterstain with DAPI solution for 10 minutes.
• Rinse briefly and mount in the antifade mounting medium.
• Analyze with a fluorescence microscope.

• Denaturation of a probe can be done in a water bath or, if available more easily in a thermocycler. The latter is more convenient, as there it is possible also to add a prehybridization step at 37°C if needed without further hand-on-time.
• 1-3 days of FISH hybridization is recommended. Stopping the incubation after 48 h may result in weaker signals while stopping after 96 h may lead to some cross-hybridization problems.

Probe

Fluorescent In Situ Hybridization (FISH) Service

Digital ISH Image Quantification and Statistical Analysis

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