Re: cryoprotection fixed brains

From:Geoff McAuliffe <mcauliff@UMDNJ.EDU>

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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HI Nancy:

    You have to freeze the tissue faster, the slow freezing in the cryostat will
engender ice crystal formation which give holes in the tissue when the crystals
melt. Freeze quickly with isopentane (2-methylbutane) cooled with liquid
nitrogen or dry ice. Even surrounding the brain with crushed dry ice is better
than putting it in the cryostat.

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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