[Histonet] "empty" cryosections!

[Favara, Cynthia (NIH/NIAID)]

Welcome to the world of histology - Gayle Callis is the expert to guide you.
If she does not respond contact her directly!

Hang in there it does get better!

x

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

Disclaimer: 
The information in this e-mail and any of its attachments is confidential
and may contain sensitive information. It should not be used by anyone who
is not the original intended recipient. If you have received this e-mail in
error please inform the sender and delete it from your mailbox or any other
storage devices. National Institute of Allergy and Infectious Diseases shall
not accept liability for any statements made that are sender's own and not
expressly made on behalf of the NIAID by one of its representatives

-----Original Message-----
From: Joanne Whitehead [mailto:joannew <@t> bluebottle.com] 
Sent: Thursday, September 29, 2005 5:38 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] "empty" cryosections!

Hi everyone,
I'm relatively new at cryosectioning, and so far have found it to be a
very frustrating experience! Some days it goes very smoothly and I get
plenty of nice sections, but most often I feel like it's a battle of
wills with the cryostat, which ususally wins.

My major problem is getting "empty" sections - the media cuts smoothly
but the tissue itself curls or crumples up, leaving just an open
circle of media. The other difficulty is with sections wrinkling as
they come off the knife, instead of lying flat. Does anyone know why
this happens, or how I can prevent it? 

I'm sectioning mouse intestinal tissue, which has been rolled into a
"Swiss roll", fixed in paraformaldehyde, infused with sucrose, snap
frozen in isopentane over liquid nitrogen and embedded in OTC. I have
tried cutting at different temperatures, from -18C to -30C, with my
samples equilibrating in the machine at least an hour before I start.


I have seen protocols in which the tissue is embedded in OTC before
freezing, and also infusing the tissue with a mixture of sucrose and
OTC before embedding. Does anyone have any preference for these
methods?

I would really appreciate any advice!
Thank you!
 
Joanne
Curie Institute, Paris




_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet