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