I would add an FC block before the first primary, I use one from Innovex for 20-30 min before the serum blocks, it is expensive but worth it for a mouse on mouse detection system.
Patsy Patsy Ruegg, HT(ASCP)QIHC IHCtech 12635 Montview Blvd. Ste.215 Aurora, CO 80045 720-859-4060 fax 720-859-4110 www.ihctech.net www.ihcrg.org -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of C.M. van der Loos Sent: Monday, September 07, 2009 1:36 AM To: anonwu...@gmail.com Cc: histonet@lists.utsouthwestern.edu Subject: SPAM-LOW: [Histonet] RE: Staining with two primary antibodies from same host Adam,Good point to prepare a protocol first, and then start staining! From theoretical point of view your protocol should work. However, I have tried this 'Jackson approach' using a Fab blocking step in a mouse-on-mouse situation (before the MOM kits were available) without success. There are at least two good solutions for double immunofluorescence using two primaries from the same species:Brouns et al. JHC 50:575-582, 2002 and Uchihara et al. JHC 51:1201-1206, 2003 applied a tyramide/fluorochrome detection for the first primary and a simple two-step for the second. Because the tyramide amplification is such a sensitive method, the first primary can be diluted up to a level that the second simple two-step detection cannot pick up signal from the first primary. Titration of the first primary antibody is most important in this procedure. You can in vitro label your goat primary with the Zenon (Invitrogen) kit based on anti-goat Fab fragments directly labeled with an Alexa fluorochrome. Next, you can built up a multistep indirect/direct double staining method:goat primary 1donkey anti-goat/fluorochrome 1normal goat serum (1:10) for blockinggoat primary 2-Zenon in vitro labeled with anti-goat Fab/fluorochrome 2lots of success with staining!ChrisChris van der Loos, PhD Dept. of Pathology Academic Medical Center M2-230 Meibergdreef 9 NL-1105 AZ Amsterdam The Netherlands Date: Fri, 4 Sep 2009 22:37:34 -0500 From: "Adam ." <anonwu...@gmail.com> Subject: [Histonet] Staining with two primary antibodies from same host To: histonet@lists.utsouthwestern.edu Hi all, I'm looking into staining with two primary antibodies from the same host, in this case goat. I've read a bit about this on Jackson Immunoresearch's website, but I wanted to run by my idea to get an idea if this is at all feasible. I want to stain mouse tissue with antigen X and antigen Y. I have a two polyclonal primaries: goat anti-mouse X and a goat anti-mouse Y. This is what I was thinking 1) Block in donkey serum for 1 hr at room temp. 2) Incubate with goat anti-mouse X overnight at 4C. Wash. 3) Incubate with Dylight 488 donkey anti-goat for 30 mins at room temp. Wash. 4) Reblock in donkey serum for 1 hr at room temp. 5) Incubate with 10 - 20 ug / mL unconjugated donkey anti-goat Fab for 1 hr at room temp -- cheapest I could find was at Rockland<http://www.rockland-inc.com/ccp8033-fab-fragment-of-affinity-purifi ed-anti-goat-igg-28-805-7102-805-7102.htm>. Wash. 6) Incubate with goat anti-mouse Y overnight at 4C. Wash. 7) Incubate with biotin donkey anti-goat for 30 mins at room temp. Wash. 8) Incubate with avidin AMCA for 30 mins at room temp Would this work? Is there an easier or better way? What are the pitalls or tips you could offer? Hope you all aren't reading this during your long weekend, Adam _______________________________________________ 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