[Histonet] RE: Staining with two primary antibodies from same host
tifei <@t> foxmail.com
Mon Sep 7 20:40:13 CDT 2009
How to label the antibody with FITC?
it is hard, or a complex procedure
------------------ Original ------------------
From: "Andrea Hooper"<anh2006 <@t> med.cornell.edu>;
Date: Tue, Sep 8, 2009 03:09 AM
To: "Histonet"<histonet <@t> lists.utsouthwestern.edu>;
Subject: [Histonet] RE: Staining with two primary antibodies from same host
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
>To: histonet <@t> lists.utsouthwestern.edu
>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
>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.
>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
>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,
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