[Histonet] Adult Mouse Skin Cryosections

[Rittman, Barry R]

Nige
It is not easy to consistently cut frozen sections of adult mouse skin.
How you orient the block and whether you have already shaved most of the
hair from the skin are critical factors. 
One reason may be that you do not have the OCT binding sufficiently to
the tissue; this can be remedied by soaking in OCT for 5 - 10 minutes
before freezing.
Theoretically you should be cutting the connective tissue side first and
the external surface with hair shafts last if possible. However if you
cut along the entire connective tissue length at one time you will
usually get some separation from the OCT. I prefer to orient the block
so that if the block is rectangular in shape, the first part to meet the
knife edge is a connective tissue corner of the block. This allows for a
gentler introduction of the tissue block and more uniformity in
sections.
Hairs will also often cause the block to cut unevenly and may result in
separation from the OCT.
As far as shaving I prefer an electric razor for my beard; however we
had a lot of problems training the mice to use this. We have found that
the best way to remove the hair from mouse skin is to use hard backed
industrial blades after the skin has been removed from the animal. These
blades are less likely to dig into the skin and cause damage than the
regular sharper razor blades. 
Barry

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Nige
Sent: Monday, November 21, 2005 4:07 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Adult Mouse Skin Cryosections

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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