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

From:"Charles Scouten"

 Freeze fracture is lines through the tissue parallel to the blade passage.  The "swiss cheese" holes are distinctly different.  

Neil, see page 48 of the January 2007 issue of Microscopy today, or the following link, for a full discussion of your problem:

Fast freezing prevents crystal formation and expansion of the water in tissue.  Expansion causes the 'swiss cheese'. 

However, the vitreous ice formed by fast freezing is an unstable state of nature.  At any temperature above something like -200C, it gradually reforms as crystaline ice.  The less cold, the faster the reformation.  Did you leave it in the cyrostat overnight to get to cutting temperature?

Long term frozen organs will gradually develop freezing artifact.  No way around it except maybe liquid helium or other outragously expensive storage.   You only solution would be cut earlier, don't store so long.  Or keep colder at all times, and cut as soon as you reach a warm enough temperature to cut.  The 30% sucrose should help some, but you are already doing that.

Charles W.  Scouten, Ph.D. 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300 x 342
FAX  314 522 0377 

-----Original Message-----
From: [] On Behalf Of David Finkelstein
Sent: Sunday, June 03, 2007 6:00 PM
Subject: [Histonet] long-term storage of rat brain at -80

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
Message-ID: <000f01c7a585$7a71f2f0$07ffc5d8@NEIL>
Content-Type: text/plain; charset=iso-8859-1

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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