Mike
Retrieval methods are also based upon the antibody. During protocol
development we try several different retrieval methods - no retrieval,
enzymatic (proteinase K), pH6 and pH9, we then determine the best method and go
from there. One retrieval method may not work for all antibodies.
Mike,
A good retrieval method will restore the epitope to a configuration that the
antibody will recognize. The data sheet received with a commercial antibody
may suggest a retrieval method to use, but if you are using a non-commercial
source, trying a variety of methods may help unless the
of my experien= ce points to antibodies that are expressed in the
nuclei may prefer high ph= AR, this is just a trend I have
noticed, definitely not something I h= ave proven.
Regards,= /font
Patsy
Original Message
Subject: [Histonet] RE
Tim-
Antigen retrieval is off-line... you need the decloaker or other device.
Brett M. Connolly, Ph.D.
Imaging Research Fellow
Merck Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
brett_conno...@merck.com
T- 215-652-2501
F- 215-993-6803
-Original Message-
From:
.
-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Morken, Tim
Sent: Tuesday, October 20, 2009 12:29 PM
To: Connolly, Brett M; histonet@lists.utsouthwestern.edu
Subject: [Histonet] RE: Antigen retrieval question
Thanks to all who replied to my question.
It is not our usual procedure to leave slides overnight in buffer after
AR and not one that we will ever repeat.
Brett
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
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,
If we need to hold retrieved slides for awhile we hold them in water up to 4
hours. Holding in water overnight leads to no signal. We have also tried
holding the slides in our Tris buffer and have found decreased or no staining.
Margaret Perry
___
hold in PBS as long as several days without any problem, under 4 C.
2009-10-22
TF
发件人： Perry, Margaret
发送时间： 2009-10-22 04:14:31
收件人： histonet@lists.utsouthwestern.edu
抄送：
主题： [Histonet] RE: Antigen retrieval question
If we need to hold retrieved slides for awhile we hold them
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.mor...@ucsfmedctr.org]
Sent: Tuesday, October 20, 2009 2:29
Perhaps the antigen is soluble and diffused out of the
tissue into the buffer?
Jerry Ricks
Research Scientist
University
of Washington
Department of Pathology
histonet@lists.utsouthwestern.edu
Perhaps reversal is not the right word...slides were left overnight in
buffer w/
: Tuesday, October 20, 2009 11:33 AM
To: Morken, Tim; histonet@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
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