Sequential FISH Procedure

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.

  1. Using forceps, carefully remove the coverslip from the previously hybridized targets.
  2. 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.

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