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
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[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
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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";
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