[Histonet] Frozen brain slices

[ANJUM PARKAR]

Hello all,
I have been having some histology issues in terms of rat brain slicing the past
few months and hence looking for suggestions to fix them, having tried most of
what I know from my past training and running of ideas real fast now. 
The following is the protocol I have used thus far:-1. Animal perfusion-a)Use
PBS first, b) Use 4%PFA next and c) Harvest brain right after PFA.
(Transcardial perfusion)2. Freeze brain by embedding in OCT compound using dry
ice and then storing at -80 until ready to slice. Alternatively some times the
-80 is used straight up with out dry ice freezing (still embedded in OCT).3.
>From standard protocol I do not do two things-a) No post fixation once brain is
harvested and b) No sucrose. Reason being, I was taught that these two steps
might result in large holes in tissue.4.For slicing, we have the older cryostat
version (Real old version of CM3050S from Vibrotome-30 micron slices) which
uses a fixed knife for slicing and has a chamber temp control and one for the
specimen. The cryostat has not been serviced for a few years now.5. In attempt
1-I take the brain out of the -80 once the chamber temperature comes to -20 and
mount the frozen mold on the chuck and attempt at slicing. Doing this I managed
to get a few slices (that too with cuts and ripples) and after which the tissue
just kept cracking and falling into pieces and hence could get no more
slices.6. In attempt 2-I thought the brain may be too cold and too hard
(perfusion and-80 freezing), so I defrosted the brain for 20 mins at room
temperature before sticking into -20 cryostat. Between slices if I felt the
tissue was too cold, I put my thumb on the specimen to warm it up. Still no
luck.7. In attempt 3-I defrosted the brain completely to room temperature, took
the brain out of the cryogel embedding (thinking that the mold was way too hard
to cut through and hence cracking the tissue too) and attached it to chuck
directly and tried slicing. Still no luck at all this time around.
>From what I have been reading online and from what I know, I primarily feel
these are issues related with temperature of the brain and that it is too cold
during cutting. I have tried different ways to bring the temperature down and I
still have no success. I have eight other brains frozen using the protocol
above and I really need to get slices out of them. Do you have any suggestions
for me? Also do you think I should make any changes to the existing freezing
protocol for the future brain harvesting?
APPenn State University,PA