[Histonet] RE: IF colocalization with 2 rabbit antibodies

Reynolds,Donna M dreynold <@t> mdanderson.org
Fri Dec 4 08:48:15 CST 2009

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From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of histonet-request <@t> lists.utsouthwestern.edu
Sent: Thursday, December 03, 2009 11:43 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 73, Issue 5
            We have been doing IF Colocaliztion with 2 rabbit antibodies since '04. Our investigators like the labeling. We do one antibody through secondary. After secondary we wash incubate with Rabbit serum 20ug/ml for 30 minutes, wash incubate with unconjugated goat F(ab)2 fragment anti-rabbit F(ab)2 fragment dilute 1:10 incubate 1-2 hr room temp. Wash then add our second primary antibody and proceed as usual. It can make a difference which  primary antibody goes first. We always try each antibody alone to see the pattern then do 2 slides rotating which antibody is first and second. Usually both ways will work but often one of the two ways is much cleaner, crisper and stronger. Good luck.

Donna Reynolds, Chief Histology Tech, HT (ASCP)
core IHC research lab
U.T. M.D. Anderson Cancer Center 
Houston, Texas

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