[Histonet] tissue processing

[Liz Chlipala]

Sheri
 
Your processing cycle is too long.  We process 20 minutes per station for mouse tissue only 2 absolutes and 2 xylenes and 3 paraffins. Even 3 absolutes and 3 xylenes at 20 minutes a station we find over processes the tissue.
 
Liz

________________________________

From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Sheri Kelemen
Sent: Wed 3/17/2010 4:05 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] tissue processing



Hi everyone,
I need some advice.
I have been doing tissue processing (on an old Shandon Hypercenter),
embedding and sectioning for 15 yrs. and never really had any problems. 
I recently processed mouse tissues on a brand new tissue processor
(Leica ASP300S). When I went to cut 5 micron sections the tissues
(spleen, heart, aorta and lung) were very dry and brittle.  They made
what looked like sawdust after each cut.  Soaking them in cold ammonia
water helped some; but they still did not cut nicely.

Here is my protocol:

I perfusion fixed the mouse with fresh 10% Neutral Buffered Formalin
(NBF).  Then I removed the tissues and cut them in smaller pieces (no
more that 5mm thick) and put them in 10% NBF for 24 hrs., then washed
with PBS and stored in 70% ethanol (made with PBS) for 2 days before
processing (only because I didn't have time to process till then).
My processing protocol is as follows:
70% ETOH     x1     1hr.
95% ETOH     x2     1hr each
100% ETOH   x3     1hr each
Xylene             x3     1hr each
wax                 x3      45 min each  60°C

Can any one tell me why they think my tissues are so so dry.  What could
I change for the next time I do this?

Thank you.

Sheri Kelemen
Research Associate
Cardiovascular Research Center, Temple University
3500 N. Broad St.
Philadelphia, PA 19140
215-707-3170 work
skelemen <@t> temple.edu

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