[Histonet] Fixing & Processing Cell Pellets

[Angela McNabola]

We used to use Histo-gel from Richard Allen, it worked quite nicely,
kept the pellet together and processed well.  It didn't interfere with
any IHC either.

I'm sorry that I can not give you more specifics as I am currently in a
new position without our protocol in from of me and do not want to give
you any misinformation, but I believe that after the fixation, we
followed the manufacturer's protocol.  Here is the link:
http://www.rallansci.com/histology/histology.aspx?id=2



Angela McNabola, MS HT(ASCP)SLS, QIHC
Research Scientist
Ikonisys, Inc.
5 Science Park
New Haven, CT 06511
203-776-0791
angela.mcnabola <@t> ikonisys.com

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Igor
Deyneko
Sent: Wednesday, April 16, 2008 10:45 AM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Fixing & Processing Cell Pellets

Dear Histonetters!
I was wondering if anyone knows or has a good protocol for processing,
fixing, and embedding the pelleted cells. I was given a protocol by my
boss,
but I have a few questions about it.
Protocol I got:

Pellet cells, wash with PBS, re-pellet.

Remove PBS and gently add 10%NBF.

10NBF-until fixed 6-24 hours.

70-85-95-95-100-100-100% Ethanols(15-20 minutes each).

3 xylene changes 20 minutes each.

4 paraffin changes 30 minutes each(melted paraffin is the first heated
step
60 degrees).

Embed and cut at 5 microns using a rotary microtome and accu edge
blades,
lay out on a 47 degree water bath, let dry on a flat warming table or in
the
oven and they are ready for deparaffinization.

So I have questions as to how would i extract cellf from the test tube
without disturbing the pellet? Then what could I put them into for
dehydration with alcohols and clearance with xylene? As far as paraffin
goes
as well,are there any special cassettes designed for cells? Also as far
as
embedding goes, would I do the standard embedding or are there special
techniques? I would appreciate any information/ protocols.

Thank you in advance.

Igor Deyneko

Infinity Pharmaceuticals,

Cambridge, MA
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