[Histonet] Curled Brain Tissue

[Gayle Callis]

Dear Alan and all,

I'm so sorry you were "flamed" as your suggestions reside in my frozen 
section file for future use. I have even passed a copy of your Histonet 
message (privately) onto people experiencing difficulties with brain.   I 
always read your comments on cryomicrotomy with "fine tuning" hints.    In 
fact, I plan to try  them IF I encounter any further problems with brain 
frozen sections.

Your expertise on the subject is greatly needed and appreciated by my 
laboratory.  The only flame I would light is in celebration of success for 
getting good ,thick frozen sections - consider it in honor of your astute, 
educated suggestions!!

Have a good weekend, and keep the suggestions coming -


At 05:25 AM 7/16/2004, you wrote:
>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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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)