Re: [Histonet] Fixation of frozen tissue?

From:Geoff McAuliffe

Hi Joe:

    Since the brain is already frozen I think you should consider 
cutting it first, mounting the sections on slides, then fixing and 
staining. Since the brain was frozen without fixation the morphology 
won't be optimal no matter what you do. Also, there may be some 
diffusion of immunoractive things. On the other hand, you could thaw  
the tissue, especially if it is too big to fit on the stage of the 
microtome, fix, cryoprotect and then cut but I don't think your results 
will be any better, perhaps worse. If you do thaw the brain, I would do 
it in cold fix so the tissue gets fixed as it thaws.
    The other thing that occurs to me is to make a 
formalin/alcohol/acetic acid fix and chill that to below freezing, and 
put the tissue in that for 24 hours, then thaw. This last idea is pure 
speculation on my part!


Joe Burdo wrote:

> I have some dry ice frozen human brain samples that I would like to
>section for IHC. We do not have a Cryostat in our lab, but we do have a
>freezing microtome. Can the frozen brain chunks be fixed in 4% PF,
>cryoprotected in 30% sucrose and then sectioned on the microtome for
>free floating IHC? I have never cut tissue that has been fixed after
>freezing, only tissue that has been fixed immediately after removal. 
>Joseph Burdo, Ph.D.
>Postdoctoral Associate
>Cellular Neurobiology Laboratory
>The Salk Institute for Biological Studies
>10010 N. Torrey Pines Rd.
>La Jolla, CA, 92037
>Histonet mailing list

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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