RE: [Histonet] Fixing & Processing Cell Pellets

From:"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:

Angela McNabola, MS HT(ASCP)SLS, QIHC
Research Scientist
Ikonisys, Inc.
5 Science Park
New Haven, CT 06511

-----Original Message-----
[] On Behalf Of Igor
Sent: Wednesday, April 16, 2008 10:45 AM
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
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
60 degrees).

Embed and cut at 5 microns using a rotary microtome and accu edge
lay out on a 47 degree water bath, let dry on a flat warming table or in
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
as well,are there any special cassettes designed for cells? Also as far
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
Histonet mailing list

Histonet mailing list

<< Previous Message | Next Message >>