[Histonet] Adult Mouse Skin Cryosections

[Nige]

Hi Gayle,

Thanks for your reply.  Yeah it is more a snap freezing method.  Is is
better for sectioning to freeze it quicker or slower?  I have been
orientating dorsal skin side on for longitudinal hair follicle sections but
have not being folding it.  With the folding technique, do you press the
hairy sides together or is it better to leave a gap between the two sides of
the tissue? The cryostat settings have been: chamber temp -30, operating
temp -20.  I've been trying 7um sections.

Many thanks,

nige

-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu] 
Sent: 21 November 2005 23:27
To: Nige; Histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Adult Mouse Skin Cryosections


Nige

How slowly are you freezing?  The freezing method you describe is not 
really a slow freeze and should work perfectly as a snap freezing 
method.  If it IS slow, then make sure the liquid nitrogen is up almost to 
top of metal block but NOT over the top of the metal block.   We remove 
mouse skin (shaved or without much hair) and folded it into a v shape so 
the hair side is on the inside of the V, and then embedded the V on edge. 
The skin orientation is such - that the knife (very sharp, disposable blade 
- changed frequently for skin) passes through the soft layers first, and 
hair side last.  If you do it the opposite direction, the layers separate 
worse as the outer layer tends to get scrunched up or pushed into the 
softer layers.   Sectioning at -20C produced excellent 5 um thick sections 
especially if the skin is inflamed.

By temperatures, which ones?  And thicknesses?

Good luck

At 03:07 PM 11/21/2005, you wrote:
>Hi,
>
>I'm having trouble sectioning adult mouse skin prepared for 
>cryosections. The dorsal skin has been taken freshly and put into 
>plastic dispomoulds with Tissue Tek O.C.T, orientated and then slowly 
>frozen by placing on a metal block which is nearly submerged in liquid 
>nitrogen.  The sample freezes nicely with no cracks, but when it comes 
>to sectioning on a cryostat the tissue tends to scrunch up and come 
>away from the tissue tek on the fatty side.  Shaving the skin had no 
>effect on the outcome. I've tried lots of different 
>temperatures/section thickness but no luck.
>
>I'm assuming the problem has something to do with the fatty layer that 
>is present with the skin, but it is very difficult (if not impossible) 
>to separate this from the skin.  Can anyone suggest how to get nice 
>adult skin cryosections, any other processing needed to be done before 
>mounting in O.C.T?
>
>Many thanks,
>
>
>nige
>
>
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)