[Histonet] Curled Brain Tissue

[Alan Bright]

I have stood back on this issue as the last time I tried to be of
assistance on this subject I was flamed, however I would not like to
deprive others of some useful information that could be of use to those
of you with difficulties in brain sectioning. They are tips that I have
posted some time ago on Histonet and I would be please to email  this
information to you. Please reply offline directly to me.


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



-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu] 
Sent: 15 July 2004 23:37
To: Rob Komorowski; Histonet <@t> lists.utsouthwestern.edu
Subject: 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, 
compression.

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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