In most cases, regardless of the cause, if inadequate FISH results are obtained, slides can be re-hybridized with a new probe mixture. If the probe is direct-labeled, then the same or a different probe can be used.
Conditions are based on peripheral blood samples.
- Using forceps, carefully remove the coverslip from the previously hybridized targets.
- Dehydrate the slides at room temperature by immersing the slide in aseries of ethanol wash solutions of 70%, 85%, and 100% for 1 minuteeach. Allow the slide to air dry. Slides are ready for repeat hybridization. It is recommended that the slide denaturation time bedecreased to approximately 50% of the original denaturation time, but not less than 3 minutes. Subsequent re-hybridization does not requirefurther reduction of the denaturation time.
The counterstain intensity decreases with sequential hybridizations, however, the loss is minimal. It is possible to perform up to 8 repeated hybridizations of the same metaphase with different probes.