- Verify the temperature of the 80ºC circulating waterbath remains constant. Fluctuation of this temperature can yield poor results. Polypropylene balls for surface insulation are available for purchase at VWR catalog # 26396-466.
To reprocess the same slide:
- Remove coverslip and put slides through Ethanol series (70, 85, 100%) each for 1 minute. Let the slides dry and put back into protease solution for 10-45 additional minutes depending on level of digestion. Do not run slides through formalin fix again. Redenature at 73ºC for 6 minutes and hybridize overnight at 37ºC. Under extreme conditions, you may need to repretreat starting at the 80ºC sodium thiocyanate and stop after protease solution-denature and hybridize again.
- If sections are not formalin fixed for 24-48 hours, proper digestion will be very difficult.
- Verify that the pH of protease buffer is 2. If not, adjust pH and enzyme will become active again.
- Make sure protease buffer is heated to 37ºC before adding protease.
- Wash paraffin sections in 2XSSC/0.3%NP-40 for 2min at 72ºC. An ambient wash following this is not recommended.
- If the pretreatment still doesn't work, you may need to switch to a more extreme pretreatment. Please contact your local Technical Service office.