1. Verify the temperature of the 80C 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:
  2. 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 73C for 6 minutes and hybridize overnight at 37C. Under extreme conditions, you may need to repretreat starting at the 80C sodium thiocyanate and stop after protease solution-denature and hybridize again.
  3. If sections are not formalin fixed for 24-48 hours, proper digestion will be very difficult.
  4. Verify that the pH of protease buffer is 2. If not, adjust pH and enzyme will become active again.
  5. Make sure protease buffer is heated to 37C before adding protease.
  6. Wash paraffin sections in 2XSSC/0.3%NP-40 for 2min at 72ºC. An ambient wash following this is not recommended.
  7. If the pretreatment still doesn't work, you may need to switch to a more extreme pretreatment. Please contact your local Technical Service office.