Multicolor FISH Protocol

GUIDELINE

Multicolor FISH (mFISH) is a method to facilitate the analysis of every single chromosome or chromosome part of a metaphase. Thus, marker chromosomes, complex chromosomal rearrangements, and all numerical aberrations can be visualized simultaneously in a single hybridization experiment.
mFISH assays can be easily completed if the reagents are purchased commercially. If the investigator plans to produce the probes and antibody mixtures in-house, many more experiments and quality-control experiments will be required.

METHODS

Prepare chromosome spreads.
Under phase-contrast microscopy, determine the extent of cytoplasmic residue on the slide preparation.
Dehydrate the slide in a 70%, 80%, and 100% ethanol series, 5 min each, and allow it to air dry.
Apply 20 µl of the RNase A working solution to the slide and coverslip. Incubate for 30 min at 37°C.
Remove the coverslip and wash the slide in 2×SSC for 5 min.
Add 10 to 15 µl of 10% pepsin to 50 ml prewarmed 0.01 M HCl and treat the slide for 5 min. Then wash the slide in 1×PBS for 5 min.
Incubate the slide in 1% formaldehyde/1×PBS/50 mM MgCl₂ for 10 min at room temperature in a well-ventilated area or fume hood.
Wash the slide in 1×PBS for 5 min at room temperature.
Pass the slide through a 70%, 80%, and 100% ethanol series, 5 min each, and allow the slide to air dry.
Denature the slide for 1.5 to 2 min in 70% formamide/2×SSC at 72°C.
Promptly place the slide into 70% ethanol following denaturation and proceed through the dehydration series.
Remove the slide from the ethanol, add the recommended amount of denatured and the pre-annealed commercial probe or 15 to 20 µl of the in-house probe, and hybridize for 48 hr at 37°C.
After 48 hr, carefully peel off rubber cement from the slide and immerse in the first Coplin jar of 50% formamide/2×SSC at 45°C. Allow the coverslip to fall off and let stand for 5 min. Remove the slide and transfer to the second Coplin jar for 5 min. Repeat with the third solution.
Wash the slide three times in three separate Coplin jars of 1×SSC, 5 min each wash, at 45°C.
Remove the coverslip and wash slide in three washes of 0.1% Tween-20/4×SSC, 5 min each wash, at 45°C with gentle agitation.
Drain excess solution, but do not allow the slide to dry, and add 40 µl of DAPI antifade counterstain. Apply coverslip and seal with clear nail polish. However, do not seal with nail polish if there are plans to re-probe the slide.
Analyze the slide using fluorescence microscopy with the appropriate filters and imaging system capable of integrating multiple fluorochromes.

Creative Bioarray Relevant Recommendations

M-FISH is defined as the simultaneous use of at least three different ligands or fluorescent dyes to specifically label DNA-excluding counterstains. Our M-FISH service provides the following 2 optional services, including whole chromosome, and specific chromosome M-FISH services.
We also are dedicated to offering a portfolio of chromosome probes for our customers to detect the amplification, deletion, and translocation of genes and improve chromosome studies, which contain whole chromosome painting probes, centromere probes, sub-telomere-specific probes, satellite enumeration probes.