Could anyone please help with this query from one of our researchers:-
I am trying to determine the best way to fix a freezing artifact. The
brains have been perfused and post fixed for a 4 weeks or more. They have
then been placed in 20% sucrose in .1M phosphate buffer until they
sink. The brains were then frozen by burying them under shaved dry
ice. Sections are to be cut in the cryostat at 40 um to preserve
I am almost positive the distortion in my cut and stained sections
is due to the freezing artifact. The artifact is less severe in smaller
brains, and is worse on samples that were frozen with dry ice pellets
rather than shaved dry ice. Most significantly, I froze one sample by
placing it above liquid nitrogen and the artifact disappeared. However,
the brain broke into many pieces.
The main problem I am having is that my specimen are large avian
brains (approximately 20 mm from anterior to posterior, 25 mm at the widest
point of the telencephalic hemispheres, and 15 mm from dorsal to
ventral). I could cut the brains in half from anterior to posterior but
the brain would will still be 12.5mm as the smallest dimension. I am
hesitant to make coronal cuts for a couple of reasons; I want to preserve a
measurement of whole brain and would lose some sections on alignment of
each specimen piece in the cryostat, the architecture of the brain has not
been mapped so trying to make the cuts in the least important areas would
be guess work. I would like to examine 4 non-localized brain regions as
well as preserve the architecture so that other researchers could examine
their own regions of interest.
If any one could suggests the best method for freezing large
specimens I would greatly appreciate it.
Mr.Laurie Reilly Ph 07 4781 4468
Physiology & Pharmacology Fax 07 4779 1526
Aust.Inst.of Tropical Vet.& Animal Sc.
James Cook University
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