[Histonet] Staining with two primary antibodies from same host

[Adam .]

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