Nancy- I hope you ARE NOT using the freezing stage in your cryostat to
freeze your tissue before sectioning.  I routinely cryoprotect fetal or
adult non-human primate brain tissue in 30%sucrose in 0.1M phosphate
buffer, place tissue pieces in molds containing OCT then freeze by placing
molds on a slab of dry ice to freeze.  Andra Erickson, UW Seattle

On Mon, 26 Mar 2001, 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,
>
> Nancy Maronto
> MPI Research
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