[Histonet] RE: Staining with two primary antibodies from same host

[C.M. van der Loos]

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 ." <anonwums1 <@t> gmail.com>
Subject: [Histonet] Staining with two primary antibodies from same
host
To: histonet <@t> 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-purified-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