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Fluorescence in situ hybridization to detect DNA sequences
FISH can also used to detect specific DNA sequences or gene loci (4, 15). To make DNA accessible to the probe, the cellular DNA must be denaturated by incubation in denaturation buffer.
Equipment and reagents
- Glass coverslips, 22x22mm
- 6-well plates
- 4% freshly made paraformaldehyde (Electron Microscopy Sciences) in PBS, pH 7.4
- Triton X-100 (Sigma)
- Parafilm
- Forceps, Dumont, GG (Electron Microscopy Sciences)
- Avidin-DCS-Texas Red or –fluorescein (Vector)
- Filterpaper
- Mounting medium (Molecular Probes)
- Microscopy coverslide
- Deionized formamide (Ambion)
Method
- Fix sub-confluent cells grown on glass coverslips in 3.7% paraformaldehyde/5% acetic acid in PBS for 15 min at room temperaturea
- Wash the cells three times with PBS for 5 min each at room temperature
- Wash the cells twice with 2x SSC for 5 min each at room temperature
- Denature coverslips in 70% formamide/2x SSC for 7 min at 85ºCb
- Prepare the probe: Denature 2 µl nick translated probe in 10 µl deionized formamide for 8 min at 95ºC
- Place immediately on ice
- Add hybridization buffer to give 50% formamide, 2x SSC, 10% dextran sulfate,1 mg/ml tRNA
- Place hybridization mixture onto each coverslip and seal with rubber cement
- Put the slide into a chamber moistened with 2x SSC and incubate for 12-16 h at 37ºC
- After hybridization wash four times in 2x SSC for 20 min each at room temperature