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

[Geoff McAuliffe]

Just a few ideas:

Do not cryoprotect unfixed tissue, it will just degrade.
Do not immerse the brain directly in isopentane. Remove brain from pup, 
mount with a dab of OCT on chuck, put stem of chuck in -150 isopentane 
cooled with liquid N2, brain will freeze in 30-60 seconds.If your chucks 
are "stemless" find a round metal (brass, aluminum) rod to put the chuck 
on while the other end in in the isopentane.
The brain may be drying out in the -80 freezer.You did not say how long 
the brains were stored before cutting.
The brain is not equilibrating in the -20 cryostat from the -80 freezer, 
ie. the brain is too cold. We always wait 30 minutes or longer.
Store sections at -80, -20 is not sufficient.
Sharpen the knife and check the angle.

Geoff

Guillermo Palchik wrote:
> 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
>
>
>
>
>
>
>
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-- 
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**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583 
mcauliff <@t> umdnj.edu
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