freezing artifact

From:Laurie Reilly

Dear All,
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.

Thank you.

Mr.Laurie Reilly                                              Ph 07 4781 4468
Physiology & Pharmacology                           Fax  07 4779  1526
Aust.Inst.of Tropical Vet.& Animal Sc.
James Cook University
Townsville  Qld. 


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