[Histonet] RE: Rat Skin

[Victoria Baker]

Hi

What Dolores has said is correct.  Zinc formalin is a good fixative for
skin.  Rodent skin has additional difficulties because of the fur and the
keratin involved with the follicles/hair.  The tissue sections should not be
more than 3 -4 mm in thickness.  Also having that  large a piece of tissue
in a cassette (length) can also contribute to some problems - not just with
fixation but with how it is placed in the cassette for processing, then
embedding and I'm sure with cutting.  My experments with rodent (rat &
mouse) had all fixation done off of the processor, on a shaker (belly dancer
platform) for twenty four hours.  Tissue was then washed in running tap
water for thirty minutes and then placed in 70% ethanol for processing.  The
ratio of solution/tissue was 20X.  No fixation was done on the processor as
it would interfere with our other protocols.  The actual processing time was
10.5 hours.  I started with 70%, 80%, 2 - 95%, 2 - abs. etoh, 50/50 -
abs/xylene, 2 - xylene and 4- changes paraffin.  I was using an older Tissue
tec processor with heat for reagents at 37 degrees C w/ vacuum and
pressure.  Watch your temperatures on the processor because if reagents
become too hot it will dry out and damage the tissue.  Paraffin was set at
65 degrees C using paraplast 2 also using vacuum and pressure.

Not knowing what your protocol may be sort of leaves me giving you only
generalities, if there is anything that I can assist you with further let me
know.

Good luck.

Vikki




On Thu, Jan 27, 2011 at 12:35 PM, Fischer, Dolores <
Dolores_Fischer <@t> baxter.com> wrote:

> The section of skin is too large. It should be no thicker than
> approximately 3mm. If you need to see the entire piece of skin, you should
> be able to submit multiple thin sections. A standard 12hr processing
> schedule works.  I am not familiar with zinc formalin, but the standard
> fixation time for formalin is, minimally 24hrs, therefore I would guess your
> fixation time is also too short.
>
> Dolores
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:
> histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Shaw, Sharon
> Sent: Thursday, January 27, 2011 10:39 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Rat Skin
>
> Can anybody share their protocol on processing rat skin I seem to be having
> a problem with it being under processed. I fix the tissue for 11hrs in zinc
> formalin and then run a 12 hour processing schedule the skin size is
> 2.5cmx1cm.
>
> Thanks,
> Sharon
>
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