Hi everyone,  

I have been having a consistent problem with my E16.5 fixed frozen brains, where when I get to the Proteinase K step the septum is destroyed.  I have used 5ug/ml for 9 minutes and have previously gotten good results but for the last two litters this has not been the case.  Only the septum is destroyed as the rest of the brain is in perfect condition.  I recently boosted the drying time of my slides from 30min to half and hour and reduced the Proteinase K concentration from 5ug/ml to 2.5ug/ml and still the septum and only the septum is destroyed.  Does anyone have any suggestions?  These brains were fixed overnight in 4%PFA.  Thank you in advance.