Re: cryoprotection fixed brains

From:Cyrla Hoffert <>

Hi, Nancy

I think your main problem is that you are freezing too slowly.  Try leaving 
the brains in your method 1 mix but with sucrose at 30%, overnight and then 
freeze it the next day on dry ice or better, in isopentane cooled on dry ice 
to -36 to -40 degrees C. Should take 45 to 60 seconds to freeze.  No longer! 
  If you don't freeze the next day, store the brains in sucrose only. You 
may already be overfixing for immuno etc. Regarding OCT, you only really 
need to use it for mounting the brain to a chuck.  It really only offers 
support for the tissue on the chuck and not for infiltration/embedding 
purposes (as far as I know!).

Hope this helps.


>From: Nancy Maronto <>
>Subject: cryoprotection fixed brains
>Date: Mon, 26 Mar 2001 22:25:58 +0000
>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 
>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. 
>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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