Digestion is performed in both the standard the new histologic FISH techniques. Digestion is required for the probe to gain access to the DNA in the nucleus. The enzyme commonly used in the new FISH technique is pepsin. In the standard FISH technique, it is protease. In general, protease is harsher than pepsin.
To perform the digestion portion of the assay:
- Remove the slides, one at a time, from the wash buffer and tap off any excess buffer.
- Using lintless (absorbent wipe) tissue, carefully wipe around the specimen to remove any remaining liquid and to keep reagents within the prescribed area.
- Organize slides and lay flat on a staining tray.
- Apply 5-8 drops (250 µL) of cold (2-8° C) pepsin to cover the specimen. ALWAYS keep pepsin at 2-8° C.
- Incubate for 10 minutes at room temperature (20-25° C). 10 minute incubation will be adequate for most specimens, but the optimal incubation time may depend on fixation and/or the thickness of the specimen. Pepsin treatment is active at room temperature for up to 50 minutes.
- Tap off PEPSIN.
- Soak sections in the diluted wash buffer for 3 minutes at room temperature (20-25° C).
- Replace the diluted wash buffer and soak sections for another 3 minutes.