Denaturation is the process by which a double-stranded nucleic acid region is separated into homologous single strands by breaking hydrogen bonds. It is an essential component of the FISH assay. Without denaturation of the probe and the DNA in the human tissue, the probe cannot bind, and signals will not be observed.
Preheat the humidified hybridization chamber to 37°C. Mark the areas to be hybridized with a diamond-tipped scribe by comparison to the H&E that has been marked for tumor areas. Verify the temperature of the denaturation solution is 72 +/- 1°C by placing a thermometer in the solution. Immerse the prepared slides (patients and controls) in the denaturing solution at 72 +/- 1°C for five minutes.
Once the tissue DNA has been denatured, the slides must be dehydrated and dried as follows:
- 70% ethanol one minute at room temperature
- 85% ethanol one minute at room temperature
- 100% ethanol one minute at room temperature
- Dry slide(s) on a 45–50°C slide warmer for 2–5 minutes
