cryoprotection fixed brains

From:Nancy Maronto <>

I need your help.  I have a project that is using 10% neutral buffered 
formalin perfused fixed brains that need to be sectioned with a crostat.  I 
have tried three methods that were suggested and found that all three 
created vacuoles in the tissue section.  The cryoprotection is not adequate. 
Paraffin sections of this tissue turns out very good.
They are:
1. 10%nbf/20% sucrose overnight in the refrigerator. The tissue sank to the 
bottom of the container.  I embedded this tissue in OCT and froze it in the 
2.  Fixed tissue infiltrated in 50% OCT overnight in the refrigerator. Next 
day embedded tissue in 100% OCT and froze in cryostat and cut sections. This 
works very well on other tissues such as livers.
3.  Three concentrations of sucrose 1 day each under refrigeration 10,20 
and30%.  Each day tissue was at bottom of container before changing 
solution.  This tissue was embedded in OCT, frozen and cut.

Method 3 turned out the best, however, not good enough. We have only a 
cryostat to use to cut this tissue.  Is it possible to cryoprotect this NBF 
fixed tissue to get good frozen sections? Any help would be appreciated.

Nancy Maronto
MPI Research
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