[Histonet] Mouse Lens cryosectioning

[patsy ruegg]

I do a lot of frozen sections on rabbit corneas and the best method I have
found includes overnight fixation in 10% NBF followed by rinses in pbs and
then infiltration overnight again in 30% sucrose, I then snap freeze the
samples and cut at about -18 as Liz suggested.  I do not get any cracks, not
done this with murine though.
Patsy

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Liz Chlipala
Sent: Tuesday, January 23, 2007 4:17 PM
To: 'David Park'; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Mouse Lens cryosectioning

David

How cold is your cryostat?  I would try to section in the warmest cryostat
as possible, possibly around -18 or so.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
P.O. Box 18592
Boulder, CO 80308
phone (303) 735-5001
fax (303) 735-3540
liz <@t> premierlab.com
www.premierlab.com
 
Ship to Address:
 
Premier Laboratory, LLC
University of Colorado at Boulder
MCDB, Room A3B40
Boulder, CO 80309

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of David Park
Sent: Tuesday, January 23, 2007 3:45 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Mouse Lens cryosectioning

Dear Histonetters,

We have been doing some cryosectioning of murine lenses for
immunohistochemistry, and have been having much difficulty in getting good
looking sections.  Of particular problem is the shattering of the lens,
especially towards the center.

We have attempted a variety of different methods, including frozen sections
that are post-fixed with methanol and acetone and previously fixing the lens
in 4% paraformaldehyde for an hour.

Are there any other kinds of fixative that you may recommend?  I have heard
of Davidson's fixative and was wondering whether this would disrupt the
actual IHC stainings.

We have also tried 4% fixation, and while the lens itself looks good when
initially cut, giant cracks begin to form over a short period of time while
the slides have been in the cryostat.  I have also tried leaving the slides
out at room temperature after doing sections to see if the shattering is
reduced, but the staining was completely gone, I think possibly because the
cytoplasm may have leaked out as a result.

Any kind of recommendation, techniques, and so forth are definitely
welcome.  Thanks.

David Park
szero1009 <@t> gmail.com
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