At 10:25 PM +0000 26/3/01, Nancy Maronto wrote:
>Hi,
>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 cryostat.
>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.
>Thanks,
>
Dear Nancy,
We perfuse with 4% paraformaldehyde, then post fix in the same 
solution for a couple of hours,then transfer to 30% sucrose (in 0.2M 
Phosphate buffer) overnight in the refrigerator or until the tissue 
sinks. Then we usually snap freeze using something similar to liquid 
nitrogen (Freeon) before embedding in OCT in the cryostat.


Cathy