[Histonet] freezing brain slices

Monfils, Paul PMonfils <@t> Lifespan.org
Fri Nov 17 10:57:50 CST 2006

I have worked out a method for freezing thin tissues flat, that works well
for me. I keep two stainless steel plates in the -80 freezer. These are
actually stainless steel covers for staining dishes. I take a thin piece of
polyethylene, about 3x3", cut from a plastic bag. Not a sandwich bag, a good
quality heavy duty plastic bag like you might use for storing specimens.  I
put a bit of OCT on the plastic, place the specimen on it, add another drop
of OCT on top, then  place a second plastic square on top. With gentle
pressure from a tongue depressor or similar flat object, I flatten and
smooth the OCT and tissue between the two pieces of plastic. Then I take it
to the freezer. I drop the plastic/OCT/tissue "sandwich" onto one of the
pre-chilled steel plates, then quickly - I emphasise quickly! - place the
other steel plate on top. The tissue is flattened, and freezes in a few
seconds.  If the tissue is soft, and I think the weight of the steel plate
might crush or compress it too much, I place a microscope slide or similar
spacer on each end of the bottom plate, place the tissue between them and
quickly put the second plate on top. That way the OCT and tissue can't be
compressed any more than the thickness of the spacer.

I then remove the OCT/tissue wafer from between the layers of plastic,
keeping it in the freezer. To mount it for sectioning without thawing, I lay
the wafer on one of the steel plates, then take a frozen blank OCT block
(frozen in the standard freezer, not the -80), soften the surface a bit with
my finger, apply a bit of additional OCT to the face of the block, then
before it can freeze, press it down onto the wafer on the cold plate.

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