To remove non-specifically bound and unbound probes, 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 solution's subsequent "cooling" when adding slides. After removing the first four slides, the solution should be allowed to return to 72 +/- 1°C before washing the following 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 required to discuss stringency and troubleshooting of FISH assays.
Following washing, slides will be air dried in the dark, and then a DAPI counterstain will be applied to stain the DNA. Slides will be cover-slipped and stored at –20°C in the dark until review. The slides will fade over time, so scoring the slides within one week is best.
