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

[Andrea Hooper]

I routinely stain with two primaries from same species. Mostly rat 
antibodies on mouse tissue. I have been fortunate enough so far not 
to have to do it in GFP containing tissue so I have utilized either 
commercially available FITc labeled primaries, or we label them 
ourselves. Then I use an anti-FITC Alexa 488 for boosting signal. 
Very nice results, some of which we have published.

Here is the general outline of the protocol (email me if you want specifics):

- Protein block
- Unconjugated primary #1 antibody (for example rat anti-X)
- Fluorophore conjugated secondary antibody (for example anti-rat IgG 
conjugated to CY3)
- Block with Rat IgG in excess to block any sites on the secondary 
which could potentially bind to your second primary antibody.
- FITC conjugated primary #2 (for example rat anti-Y-FITC labeled)
- Alexa 488 conjugated anti-FITC

X protein will be labeled red
Y protein will be labeled green




>
>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
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