[Histonet] Flash Freezing Brain

[David Finkelstein]

Dear acjanes <@t> bu.edu,

Your poor brains!!  ;)
Holes in frozen tissue = freeze fracture.
Never store brains at -20 only at -80c.
I agree with the other comments about how to freeze onto the chuck with Oct.

For long term storage of fixed brain (remember the brain will continue to
slowly fix in the freezer, so the time depends on the antigen).  
1) place brain on small piece of foil or piece of plastic weigh boat.
2) place a separate piece of foil with label on dry ice.  
3) lower the brain into the vapours of the liquid nitrogen just\on or above
the liquid level.  Use long plastic forceps.  You don't wont to freeze it
too long or too quickly or it will crack.
4) quickly transfer the brain onto the  foil in dry ice.  Do not let the
brain warm up by touching it with your fingers.
5) drop foiled brain in a plastic bag, place immediately onto dry ice and
then -80 freezer.

Assoc. Professor David Finkelstein,
The Mental Health Research Institute of Victoria,
155 Oak Street, Parkville, Victoria 3052 
AUSTRALIA 
Mobile: 0409171227
Tel: +61 (03) 9388 1633 
Fax: +61 (03) 9387 5061
dfinkelstein <@t> mhri.edu.au

[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
acjanes <@t> bu.edu
Sent: Monday, October 23, 2006 1:00 PM
To: histonet <@t> lists.utsouthwestern.edu
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.