[Histonet] Issues regarding flash freezing and cutting of Rat pup brains

[Guillermo Palchik]

Dear Histoneters,

I am looking for help regarding flash freezing of rat pup brains  
(Postnatal day 8). We need the tissue to be fresh (unfixed) for  
cutting at the cryostat.
So far the technique has been:

1-  to scoop the brains straight into cold (-50 C) Isopentane for  
about 10 seconds,
2 - dry for 10 seconds in dry ice,
3- wrap in tinfoil and into the -80 Freezer it goes.

When it's time to cut, we apply a dab of OCT, flatten it with the heat  
extractor, apply another dab of OCT and stand the brain on the  
cerebellum (olfactory bulbs facing the person when cutting) and do a  
"ring" of OCT around the cerebellum (so actually most of the brain  
hits the cryostat blade directly (temp of cryostat ~ -20C). Mount on  
slides and put in the slide warmer (~ 37 - 40 C for 15 min). The  
slides then are all placed in a box and  stored into a -20C freezer  
until processed for TUNEL (usually 3-5 days). As I said, the tissue  
does not look good (it curls, it cracks, etc..).

I wanted to try mounting the whole brain into OCT and flash freeze the  
OCT block, and also even cryoprotecting the brains beforehand. I  
wanted to ask around if it is a good idea to cryoprotect (30% sucrose,  
O/N) since the brains are not fixed....
My "assumption" is that since the brain has not been fixed/perfused,  
upon bringing it up to room temperature, (or even 4C), enzymatic  
activity would resume and degrade the tissue, but I could be wrong...
Any / other advice will be GREATLY appreciated, as well as pointing  
out gross mistakes on our part...
Protocols also accepted!

Thanks a lot.

Gil Palchik
gp62 <@t> georgetown.edu