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 ." <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-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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