Standard Post-Hybridization and Completion

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Standard Post-Hybridization and Completion

To remove non-specifically bound and unbound probe, the slides will be washed in the post-hybridization wash solution at 72 +/- 1° C for two minutes. The post-hybridization wash used for paraffin based tissue slides is 2X saline-sodium citrate (SSC)/0.3% NP-40.
Temperature is a critical component. Only four slides should be washed at a time due to the subsequent "cooling" of the solution when adding slides. After removing the first four slides, the solution should be allowed to return to 72 +/- 1° C before washing the next four slides.
Other post-hybridization washes can be used depending on the stringency level needed and the complexity of the testing, but this discussion is beyond the scope of this course. An advanced course will be needed to discuss stringency and troubleshooting of FISH assays.
Following washing, slides will be air dried in the dark and then DAPI counterstain will be applied for staining of the DNA. Slides will be cover-slipped and stored at –20° C in the dark until review. The slides will fade over time, so it is best to score the slides within one week.