[Histonet] long-term storage of rat brain at -80

From:"David Finkelstein"

Dear Neil 
It sounds like freeze fracture not a storage problem. Long tem freezing
causes freeze drying not holes.
Two suggestions.
1) Get rid  the OCT.  Ether suspend the brain in the mold just above the
liquid nitrogen or drop the brain into the  isopentane for no more than 20
sec. Then place the brain on dry ice. Keep the plastic bags on dry ice. Wrap
the brains plastic file kept on dry ice.  Then put them in the bags.  Always
transport them in or on dry ice.
  2) use metal forceps. Keep the tips in dry ice.
Good luck

Assoc. Professor David Finkelstein,
The Mental Health Research Institute of Victoria, 
155 Oak Street, Parkville, Victoria 3052 AUSTRALIA

Message: 4
Date: Sat, 02 Jun 2007 20:18:30 -0600
From: Neil Fournier 
Subject: [Histonet] long-term storage of rat brain at -80
To: histonet@lists.utsouthwestern.edu
Message-ID: <000f01c7a585$7a71f2f0$07ffc5d8@NEIL>
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I thought many of you might able to help me devise a protocol for long-term
storage of flash-frozen perfused rat brains.  I am sure what I am doing
currently is not appropriate.  Our procedure thus far is to perfuse rats
with saline followed by 4% paraf
ormaldehyde. Post-fix for 48 h and then cryoprotect in 30% sucrose until the
brain sinks.  The brains are then removed from sucrose and dried with a Kim
Wipe, blocked in the coronal plane, and placed in cryomolds containing OCT.
The mold is then frozen
 in liquid nitrogen cooled isopentane.  The frozen mold is then placed in a
empty ziplock bag and placed in the -80 freezer.  Several months after I did
this procedure and sectioned the brains, I found significant holes
indicative of freezing artifact i
n the tissue.   Although I have had some problems in the past with my
isopentane freezing protocol,  I have a feeling that this issue is probably
the result of storage at -80 degree C but I cannot be for certain.  (I do
not believe the issue is perfusio
n based si

Could anyone share with me their protocol or provide suggestions?  Lastly, I
have looked high and low but does anyone know where I can find some long
plastic forceps that would be of adequate length for flash freezing?  Our
current one was broken accide
ntally and none of us know where it came from although we are now beginning
to suspect that there might be a mystical histofairy who is responsible for
the often magically appearing (and sometimes disappearing) histological
chemicals and equipment in la

Thanks again,



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