[Histonet] croystat sectioning
I have a problem with cryostat sectioning that is driving me crazy. I hope someone out there can help because we have exhausted our brains coming up with solutions that have not helped. I am sectioning perfused rat brain on a cryostat, 20u thick, for IHC on floating sections. I have been doing this successfully for years.
Now the problem is that they are curling up into tight little rolls in the PBS, which make them impossible to stain. They even curl as soon as they come off the knife edge, so I can't even put them on slides.
We have: tried several different formulas for the fix, 3 different people doing the perfusion, 3 different kinds of PBS, warm PBS, cold PBS, all possible knife angles, different knives, different side of the knife, made the block colder, or warmer, thicker sections, thinner sections,
cut fast, cut slow, NOTHING HELPS!
When we cut fresh frozen brain to put on slides we do not have this problem. So, naturally we think it is the fix, or the method, or the person doing the perfusion. So, I had the expert do a perfusion and it still happened. At this point I just wanted to go out and get drunk and I am not a drinking person.
Can anyone help? All advice is appreciated.
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