[Histonet] RE: Antigen retrieval question

[Mejia, Maria]

Brett,

We mostly work & do IHC on 40um free-floating brain sections.  These sections were cut from fixed brain blocks.  Sometimes, not on
a regular basis, but sometimes we have had to place our sections in (plain) PBS solution & stored at 4C - overnight before continuing the IHC
protocol with no effect to the staining quality.  Keep in mind, that this was done before the primary antibody. We have also placed our cut
and unstained sections in plate wells (5ml of PBS) - again at 4C for 2 days before starting our IHC protocol.  We use quite a number of 
different antibodies & stain using polymer.

I hope this helps.

Maria Mejia
Histology Manager
Department of Neurosurgery
UCSF
Sf, CA 94103
 
________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Connolly, Brett M [brett_connolly <@t> merck.com]
Sent: Tuesday, October 20, 2009 11:33 AM
To: Morken, Tim; histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: Antigen retrieval question

Perhaps reversal is not the right word...slides were left overnight in
buffer w/ tween. We got very minimal faint staining compared to previous
runs done in one day.

Brett

-----Original Message-----
From: Morken, Tim [mailto:Timothy.Morken <@t> ucsfmedctr.org]
Sent: Tuesday, October 20, 2009 2:29 PM
To: Connolly, Brett M; histonet <@t> lists.utsouthwestern.edu
Subject: RE: Antigen retrieval question

Brett, how long? I've left them overnight before proceeding without any
problems.

It would not be "re-fixation" as with formalin. However, if it is in a
detergent buffer it might cause some other kind of denaturation.


Tim Morken
Supervisor, Histology / IPOX
UCSF Medical Center
San Francisco, CA


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
Connolly, Brett M
Sent: Tuesday, October 20, 2009 11:22 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Antigen retrieval question

Has anyone experienced a "reversal" of citrate HEIR unmasking do to a
prolonged delay in continuing the experiment after the retrieval step?

Brett M. Connolly, Ph.D.
Research Fellow, Imaging Dept.
Merck & Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
tel. 215-652-2501 fax. 215-993-6803
brett_connolly <@t> merck.com


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