Re: brain frozen sections

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From:Geoff McAuliffe <mcauliff@UMDNJ.EDU>
To:"Robert G. Russell" <>
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"Robert G. Russell" wrote:

> Histonetters,
> this is a follow up re our probelms with artefacts in frozen brain
> sections.  We are now getting good sections after sectioning with no
> artefacts at that time.  However the investigator brought back the sections
> a few days later, after storage at ?$ c in refrigerator, now there are
> small holes all over, especially in the cortex.  Not bad freeze thaw
> artefacts, but diffuse enought to affect sensitive immunostaining for
> specific cell types. Brains were fixed in 4% paraformaldehyde, the to PBS
> then to 30% sucrose, frozen in OCT boat in Shandon histobath and direct to
> cryostat.  The OCT around the sections now appears wrinkled and has "water"
> hole artefacts similar to the tissues.  The brains are from mice infected
> with virus.  The problem does not appear to be
> preparation/embedding/sectioning related. Any suggestions?.  Is the sucrose
> step causing osmotic effects related to the section:OCT gel interface,
> hence resulting in subsequent "holes".  Any other insights? Is there a
> different embedding/support medium we could use v's OCT?
> Bob
> Dr Robert G. Russell
> Director
> Histotechnology and Comparative Pathology

    Assuming that the virus is not the cause of the holes (spongiform
encephalopathy), you are freezing too slowly. This will allow large ice
crystals to form. When they melt, one gets holes! I don't know about freezing
in a "Shandon Histobath", I mount the brain on a freezing stage (which freezes
from below) and I surround the brain with crushed dry ice. Alternativly, if you
are using a cryostat, mount the brain on a chuck with a bit of OCT to keep it
in place. Then put the chuck, not the brain, in isopentane (2-methylbutane)
that has been cooled with liquid nitrogen (or dry ice). I do not think it is
necessary to "embedd" the brain in OCT.

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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