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Dehydrate the slides at room temperature by immersing the slide in a series of ethanol wash solutions of 70%, 85%, and 100% for 2 minutes each. Allow the slide to air dry. Slides are ready for FISH. It is recommended that the slide denaturation time be decreased to approximately 50% of the original denaturation time, but not less than 3 minutes. Subsequent re-hybridization does not require further reduction of the denaturation time.
The following steps do not remove 100% of the H&E stain (which appears as autofluorescence on the slide); however, most slides should be interpretable when these steps are followed.
To make 50% PEG:
Melt the PEG in a waterbath or incubator 50-75C, then dilute with purified water, 100g in 100 mL.
To make 1 Liter of Carbowax:
Milli-Q water: 435 mL
PEG 1500, 50%: 40 mL
(PEG = polyethylene glycol Catalog #P5402 from Sigma-Aldrich)
Ethanol, 95%: 525 mL Expiration: 1 year
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.
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.