Re: cryoprotection fixed brains

We have cut previously NBF fixed tissue  on the cryostat using the "Gentle
Jane" system and have gotten very good sections.  I do not remember if we
did brain but rat and mouse kidney, liver, spleen all cut well.
Gina Sennello
Gilead Sciences

                    Nancy Maronto                                                           
                    <nmaronto@hot        To:               
          >            cc:                                                
                                         Subject:     cryoprotection fixed brains           
                    03:25 PM                                                                

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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