RE: [Histonet] RE: Antigen retrieval
The most important part= about working up a new antibody is to have known positive tissue to use as= a control, if you do not have that everything you do is a shot in the dark= , you will not know if your protocol is not working or the control you are = using is not expressing that antigen. Each antibody has it's own requ= irement for pretreatment or not. We do what Liz does and sometimes ad= d in a ph8 buffer when nothing else works. Just to save on slides to = run we start with no ar and ph6 and if we do not get good results, go to ed ta ph9, then PK, then ph8, if all these fail, we revert to overnight incuba= tion of the primary ab after the most agressive high ph buffers and/or enzy= me digestion. If that fails we give up. This is all of course a= fter the best antibody titer has been determined. Some 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: Antigen retrieval From: Elizabeth Chlipal= a <[1]l...@premierlab.com> = Date: Wed, June 13, 2012 7:32 am To: 'Mike Tighe' <[2]mti...@trudeauinstitute.org>, = "[3]histo...@lists.uts= outhwestern.edu ([4]h isto...@lists.utsouthwestern.edu)" <[5]histonet@lists.utsouthwestern.edu> 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. Liz Elizabeth A. Chlipala, BS, HTL(AS= CP)QIHC Manager Premier Laboratory, LLC PO Box 18592 = Boulder, CO 80308-1592 (303) 682-3949 office (303) 682-9060 = fax (303) 881-0763 cell [6]w= ww.premierlab.com Ship to address: 1567 Skywa= y Drive, Unit E Longmont, CO 80504 -Original Message= - From: [7]histonet-boun...@lists.utsouthwestern.edu [[8]mailto:histonet-bounces@lists.utsouthw= estern.edu] On Behalf Of Mike Tighe Sent: Wednesday, June 13, 201= 2 8:27 AM To: [9]hi= sto...@lists.utsouthwestern.edu ([10]histonet@lists.utsouthwestern.edu) Subject: [Histo= net] Antigen retrieval Does anyone have a favorite antigen ret= rieval method for FFPE mouse tissues that they would be willing to share? I= have been using citrate buffer Ph6.0 with poor to moderate results. Thanks= for any help! Mike __= _ Histonet mailing list [11]Histonet@lists.utsouthwestern.edu [12]http://lists.utsouthwestern.edu/mailman/listinfo/histonet = ___ Histonet mailing l= ist [13]Histonet@lis= ts.utsouthwestern.edu [14]http://lists.utsouthwestern.edu/mailman/listinfo= /histonet References 1. 3D"mailto:l...@premierlab.com"; 2. file://localhost/tmp/3D"m 3. 3D"mailto:histonet@lists.utsouthwestern.edu"; 4. 3D"mailto:histonet@lists.utsouthwestern.edu"; 5. 3D"mailto:histonet 6. 3D"http://www.premierlab.com"/ 7. 3D"mailto:histonet-bounces@lists.utsouthwestern.e 8. 3D"mailto:histon 9. 3D"mailto:histonet@lists.utsouthwestern.edu"; 10. 3D"mailto:histonet@lists.utsou 11. file://localhost/tmp/3D"mail 12. 3D"http://lists.utsouthwestern.edu/mailman/listinfo/histonet 13. 3D"mailto:Histonet@lists.utsouthwestern.edu"; 14. 3D"http://lists.utsouthwestern.edu/___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Antigen retrieval
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 epitope is irreversibly altered by fixation. We run all new assays using pH 9, pH 6 and sometimes pH 3 [10 min treatment rather than 20 min] in addition to enzymatic retrieval using Pepsin, Trypsin or Protease and finally No Retrieval. Happy Hunting, Tresa ___ Does anyone have a favorite antigen retrieval method for FFPE mouse tissues that they would be willing to share? I have been using citrate buffer Ph6.0 with poor to moderate results. Thanks for any help! Mike ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Antigen retrieval
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. Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308-1592 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell www.premierlab.com Ship to address: 1567 Skyway Drive, Unit E Longmont, CO 80504 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mike Tighe Sent: Wednesday, June 13, 2012 8:27 AM To: histonet@lists.utsouthwestern.edu (histonet@lists.utsouthwestern.edu) Subject: [Histonet] Antigen retrieval Does anyone have a favorite antigen retrieval method for FFPE mouse tissues that they would be willing to share? I have been using citrate buffer Ph6.0 with poor to moderate results. Thanks for any help! Mike ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: antigen retrieval
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: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Truscott, Tom Sent: Wednesday, March 21, 2012 4:28 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] antigen retrieval To those with the Biocare intelliPATH system, Is the antigen retrieval part of the IHC automated or do you need to use the Biocare decloaker? Thankyou, Tom Truscott ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] RE: Antigen retrieval question
Yes, I have, redoing the AR does fix that though.
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
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-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
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-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Connolly,
Brett M
Sent: Tuesday, October 20, 2009 11:22 AM
To: histonet@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_conno...@merck.com
Notice: This e-mail message, together with any attachments, contains
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Re: [Histonet] RE: Antigen retrieval question
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 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Antigen retrieval question
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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] 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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Connolly, Brett M Sent: Tuesday, October 20, 2009 11:22 AM To: histonet@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_conno...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [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 fax. 215-993-6803 brett_conno...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Antigen retrieval question
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-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Connolly, Brett M [brett_conno...@merck.com] Sent: 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 day. Brett -Original Message- From: Morken, Tim [mailto:timothy.mor...@ucsfmedctr.org] Sent: Tuesday, October 20, 2009 2:29 PM To: Connolly, Brett M; histonet@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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Connolly, Brett M Sent: Tuesday, October 20, 2009 11:22 AM To: histonet@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_conno...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Antigen retrieval question
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/ 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 PM To: Connolly, Brett M; histonet@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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Connolly, Brett M Sent: Tuesday, October 20, 2009 11:22 AM To: histonet@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_conno...@merck.com _ Hotmail: Trusted email with powerful SPAM protection. http://clk.atdmt.com/GBL/go/177141665/direct/01/___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[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.mor...@ucsfmedctr.org] Sent: Tuesday, October 20, 2009 2:29 PM To: Connolly, Brett M; histonet@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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Connolly, Brett M Sent: Tuesday, October 20, 2009 11:22 AM To: histonet@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_conno...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
