[Histonet] freezing brain slices

[Alan Bright]

I go along with this method.

Best Regards

Alan Bright

Bright Instrument Co.Ltd.
St Margaret's Way
Huntingdon
Cambridgeshire
PE29 6EU
England

Tel No:+44 (0)1480 454528
Fax No:+44 (0)1480 456031
Email: abright <@t> brightinstruments.com
Web Site: www.brightinstruments.com
Skype User ID: dazzle0


-----Original Message-----
From: Monfils, Paul [mailto:PMonfils <@t> Lifespan.org] 
Sent: 17 November 2006 16:58
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: RE: [Histonet] freezing brain slices


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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