Can anyone help with this problem?   We routinely fix mouse brains for
immunohistochemistry and in situ work.  Brains are removed from mice
very fast , divided sagitally into hemibrains and fixed in 3%
paraformaldehyde for up to 5 hours.   Then embedding in OCT and freezing
in molds happens.  We sometimes, not always, find that
the centers of the sections are not as well preserved as the marginal
zones.   Is this a fixative delivery problem or a cryoprotection
problem?  The hemibrain division should ensure that tthe fixative
penetrates the center of the tissue in 5 hours.  But the OCT almost
certainly does not.  Should we be using other cryoprotectants?  DMSO,
glycerol, sucrose?   Should we further subdivide before fixation?  Any
recommendations.   We have ruled out perfusion fixation because delivery
of fixative is too slow and mostly via capillaries which clog very
easily.  We can get the fixative to the brain tissue faster by rapid
removal of brain, division and immersion.  Under 4 minutes.  Brain
perfusion takes ages and I am not convinced that some areas ever "see"
fixative.
Thanks
David Sanan
Gladstone Institutes, San Francisco