[Histonet] Intestinal Tissue Questions

[Rittman, Barry]

Hi
Here in the lone star state we generally say  y'all to an individual or
y'all y'all to a group.
Hi to y'all in Wisconsin - we lived in Iowa for several years and
enjoyed visits to several wonderful places in your state.

1.	My suggestion is that once you have fixed in buffered formalin
is to store in 70% ethanol. If for a longer time then you may wish to
consider adding 10-20% glycerin. This is expensive but guarantees that
tissue will never dry out. Only reason not to use 70% is if you wish to
do frozens for lipids some time later as some lipids will be lost.

2.	Most mucosae will tend to curl in this manner. What I would
recommend is to pin the tissue on a piece of cork and then place tissue
side down in the fixative. Must be careful to use stainless steel or
plastic pins as some regular steel pins will rust onto the tissue. (we
used to use small hedgehog quills for this purpose but these are not
readily available here).
Once tissue is fixed then it should not curl up and can be processed
normally. It does not take long for tissue to become hardened
sufficiently to allow you to remove from the cork so that fixation can
be completed after removal.
If you have really small pieces can place the fresh tissue on a piece of
thin card such as postcard with the connective tissue side down for
fixing. The tissue fluids will bind the gut to the card.  
Would recommend removal of card prior to sectioning!
Hope this helps
Barry

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
wbedale <@t> biochem.wisc.edu
Sent: Friday, January 21, 2005 10:32 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Intestinal Tissue Questions

Hi to all of you,

I'm quite new to histology and have a few basic questions.

Here's what I'm doing:

Tissue:  mouse intestine;  small and large intestine is harvested, cut
open longitudinally, and rinsed with PBS prior to fixation; 3 cm lengths
are fixed.
Fixation: 10% formalin
Processing and embedding:  manual processing, paraffin embedding, 4-6
micron sections
Staining:  H&E staining

Two questions:

1.  After fixation, is it better to store my tissue (for ~1 month or so)
in fixative, or in 70% ethanol?

2.  I'm attempting to get transverse sections of the intestine;
sometimes
I notice that my tissue ends up inside out (mucosa facing outward).  I
think it may happen when I use a razor blade to cut the tissue
longitudinally vs. using scissors to make the longitudinal incision, and
am going to test that.  Has anyone had any experience with this?

Thanks for any advice.

Wendy

Wendy Bedale, Ph.D.
Assistant Scientist
Department of Biochemistry
University of Wisconsin-Madison
433 Babcock Dr.
Madison, WI 53706
608-262-3099 ext. 3266
wbedale <@t> biochem.wisc.edu


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