[Histonet] Unsuccessful Muscle Lipid Staining

[Liz Chlipala]

Son

We did this in the past, we post fixed in osmium, it turned out quite =
nice.  I'll send images in a different e-mail.  We fixed in formalin and =
then post fixed the muscle in osmium overnight, we were working on mouse =
tibialis anterior so we kept the entire muscle intact and processed the =
whole muscle and then cut the muscle in half prior to embedding.  We =
found that the unstained sections turned out the best for demonstration =
of the fat. I might have a material and methods some where since I think =
they published on it.  I'll send that to you with the images.

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
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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 Sonny =
Duong
Sent: Sunday, July 22, 2007 3:31 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Unsuccessful Muscle Lipid Staining

Hi all,

I have been trying to stain mouse muscle tissue for intramuscular =
triglyceride droplets with Oil Red O (dissolved in triethyl phosphate) =
for some time now, and my results have been disastrous.  I've tried it =
on both fresh frozen and formaldehyde fixed tissues (and cryoprotected) =
cryosections, but almost always it seems that the lipid droplets within =
the fibers (maybe?) leak out of the cells and conglomerate on the edges =
of the tissue and within gaps between the cells.  Very few of the =
fibers, if any, display any lipid droplet staining and it is always very =
weak. The lipid droplets all conglomerate in roughly the same positions =
between serial sections as well, which leads me to believe something is =
happening during the freezing/fixation process, or the cutting in the =
cryosection.  Does anyone have any suggestion as to what I could be =
doing wrong here (i.e cutting technique, temperature, fixatives)?  Also, =
does anyone have any experience with using ORO in triethyl phosphate?

Thank you all for your time,
Sonny Duong
University of Virginia
Green Lab



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