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

[Merced M Leiker]

Nice! I may try one or both of your procedures (Adam & Andrea).

~Merced

--On Monday, September 07, 2009 3:09 PM -0400 Andrea Hooper 
<anh2006 <@t> med.cornell.edu> wrote:

> 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-pu
>> rified-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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>
>
> --
>
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Merced M Leiker
Research Technician II
Cardiovascular Medicine
348 Biomedical Research Building
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214  USA
leiker <@t> buffalo.edu
716-829-6118 (Ph)
716-829-2665 (Fx)

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