Re: [Histonet] Curled Brain Tissue
We just set up hamster brain perfused with Periodate Lysine
Paraformaldehyde, cut into coronal slices after a few hours immersion in
PLP. Slices were slices cryoprotected overnight in 25% sucrose in
PBS. Snap freezing was done using empty petri dish floating on liquid
nitrogen inside styrofoam box. The slice of brain (coronal) was embedded
in OCT in Tissue Tek mold and allowed to freeze inside extremely cold petri
dish. One piece of tissue per block.
Cryosectioning was done with Leica 1850 using that model's disposable knife
holder with Accuedge high profile blade and the glass anti-roll
device. Knife angle was set at 12 degrees. Temperature at knife, sample
and anti-roll plate was -19C, micrometer set at 50 um. Antiroll plate was
cleaned with alcohol and dried before sectioning. Block was oriented so it
looked like a diamond shape, cutting on a point was path of least
resistance and for capture with brush when necessary.
The section passed under roll plate smoothly, without compression, totally
flat until it hit warmer area of antiroll plate. As section finishes
coming off block, it does begin to curl back towards knife edge. This
problem was solved by capturing end of curled OCT area with a brush (long,
thin, fine bristles), and holding section to keep it from curling
while antiroll plate was raised. With careful manipulation, the section
was picked up onto slide (lowering slide to section). Another brush was
employed to hold top of section if it was needed. At no time did the brush
touch tissue, only OCT - making it easy to play with the section. Also
tried some "heavy breathing" onto section which helped it stay flat until
slide pickup time, just don't melt the section!
Another technic was tried - after capturing section to keep it from curling
up, the section was slid onto top of a totally cold Plus charge slide (had
been stored in cryostat). After section was on slide, I slide was warmed
on back of hand - section melted onto glass. This was more difficult with
thick 50 um section than with a thin 5 um section.
It was easier to reach under anti roll plate to keep section from curling
rather than slide section onto cold slide and melt it. These onery thicker
sections just loved to curl up!
Sectioning was done in a slow, steady motion.
Evaluation of situation: The section tends to warm up the further it
travels under the antiroll plate, causing it to curl. With patience and if
one can capture the curl to prevent a major curl up, then you have a
chance. No sticking to the antiroll plate was experienced nor wrinkling,
We will be trying the dry ice cooling of anti roll plate to see if this
solves some of curling problem. Alan Bright gave this helpful, clever hint
some time ago. I also wonder if touching the metal plate of knife holder
with dry ice will help, anything to keep the warming trend to a minimum.
It was impressive to see this anti roll plate work so well, as I am
normally a "brush" person. I tried brush but with thicker sections it take
a stronger set of bristles to flatten/prevent curling.
Sorry for the long description of what was done, but maybe the fine detail
will help -
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