FW: [Histonet] "empty" cryosections!

[Jasper, Thomas G.]

Thomas Jasper HT(ASCP)BAS
Anatomic Pathology Coordinator
SMDC Clinical Laboratory
Duluth, MN
tjasper <@t> smdc.org


-----Original Message-----
From: Jasper, Thomas G. 
Sent: Thursday, September 29, 2005 11:03 AM
To: 'Favara, Cynthia (NIH/NIAID)'; ',histonet <@t> lists.utsouthwestern.edu'
Subject: RE: [Histonet] "empty" cryosections!


Dear Joanne,

One possible obstacle to optimal frozen tissue sectioning may be the
fixation step prior to freezing.  I have not used paraformaldehyde to fix
and then freeze tissue for cryosectioning.  However, I have been asked to
cut frozen sections on tissue that has been formalin fixed.  The degree of
difficulty seems to increase the longer the tissue has been in a fixative.
In other words, if we get it out of formalin quickly (within minutes) we'll
probably get a decent section.  If half the day has gone by we probably
wouldn't try.  Once a section is obtained the main problem is usually one of
adherence.
This experience is in a clinical setting, with human tissue, which leads me
to my next point.
Generally speaking, animal tissue is much drier, and therefore more
difficult to section period.  Also, if you are getting empty sections (no
tissue) you may be having a problem with your supporting media (OCT, etc.).
If the media is not adhering well to the tissue, it would lend itself to
creating these holes.  Fatty tissue is difficult to freeze as well, although
I note that you have tried sectioning at different temperatures.  I believe
I have seen postings from others to drop the temperatures very low to help
freeze the fatty tissues.  I know there are others monitoring the Histonet
with more knowledge about frozen sectioning.  I just thought I'd share some
of my experiences, hopefully it will be of some help.  Good luck to you!
tj

Thomas Jasper HT(ASCP)BAS
Anatomic Pathology Coordinator
SMDC Clinical Laboratory
Duluth, MN
tjasper <@t> smdc.org


-----Original Message-----
From: Favara, Cynthia (NIH/NIAID) [mailto:cfavara <@t> niaid.nih.gov]
Sent: Thursday, September 29, 2005 9:47 AM
To: 'Joanne Whitehead'; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] "empty" cryosections!


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

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-----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




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