RE: [Histonet] Flash Freezing Brain

From:"Charles Scouten"

The holes are commonly referred to as "Swiss Cheese Artifact" and are
due to freezing too slowly.  Try freezing the brains directly by
immersion in isopentane, no foil, no OCT.  You may be using so much OCT
it is slowing down the rate of heat transfer. 

Then put just a little OCT on the pedastal as glue, put the block of
tissue on, and freeze again. 

How long do you store the tissue?  Eventually, the ice reformats, and
you get Swiss Cheese no matter how quickly you froze it, or no matter
how cold you stored it.  It depends on a lot of variables, but I would
give you a month.  I have heard stories of a year.

See the article in the following link:

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

-----Original Message-----
[] On Behalf Of
Sent: Monday, October 23, 2006 1:00 PM
Subject: [Histonet] Flash Freezing Brain

I have a question about flash freezing rodent brains, as sometimes I see
little holes destroying my tissue and I cannot determine the cause.

Normally I perfuse mice with 4% para, post fix for 1-4 hours with para
at room temp then put in 30% sucrose until the brain drops (At 4C). 
After this I make an foil mold which I fill with OCT and put my brain
inside. I then slowly lower the foil/brain mold into a bath containing
isopentane and dry ice just until everything freezes. I then store the
tissue at -20 until ready to cut. Normally this works, but occasionally
I loose tissue to the holes. I am now going to be starting a project
using rats and I want to make sure all the tissue stays intact. Does
anyone have suggestions?

We need the tissue to run ICC for c-fos the brains will be cut at 40
microns using a cryostat.

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