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
g...@georgetown.edu
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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
mcaul...@umdnj.edu
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