I would strongly recommend against trying to use primary and secondary antibodies raised in the same species in double labeling experiments like you propose. You are just begging for false positives!  Even if you image each channel seperately and your red channel is clean,  you will still have false-positive Alexa488 signal present in your green image. When you overlay the two images to assess the localization of the antigens, you will not be able to distinguish what double labeling is real and what is false.

The Donkey anti-goat Alexa596 secondary antibody will bind to BOTH your Goat anti-Y and your Goat anti-Mouse-Alexa488. This will give you some false positive labeling. Standard secondary antibodies have 2 antigen binding sites on them. When the Donkey anti-Goat Alexa596 binds to Goat anti-Y,  it will still have one of its binding sites available to bind to another Goat IgG.  This second unoccupied binding site will be available to bind to your Goat anti-mouse Alexa488.  This will create a complex of Donkey anti-Goat 596 PLUS Goat anti-mouse Alexa488 bound onto your Goat anti-Y primary, producing false-positive double labeling.

I can think of three potential solutions. Options 1 and 2 are the better choices:

1)   The best option would be to get an Anti-Y or an Anti-X primary antibody from a non-goat species for your experiment if such antibodies are available.

2)   Get an anti-mouse Alexa488 secondary antibody raised in some species other than goat (or sheep as most anti-goat secondary antibodies will happily bind to sheep Igs as well). There are many host options available these days-- rabbit, chicken, hamster, etc....  If you choose a secondary antibody raised in a rodent (rat, hamster, etc) you could experience cross-reactivity with the mouse Alexa488 secondary. Rabbit may be the best bet here.

3)    You might be able to dodge the unwanted binding between the Donkey anti-goat Alexa596 and the Goat anti-mouse Alexa488 antibodies problem by following the sequential labeling procedure suggested above by Stefan but substitute an Fab-fragment Donkey anti-goat Alexa596 secondary antibody for the standard Donkey anti-Goat Alexa596 antibody that you have. The Fab fragment has only one binding site on it, and once bound to the Goat anti-Y it would not be able to bind to the Goat anti-mouse Alexa488.

Hope this helps. Good luck.

Dave Sherry

Hy,

it can be so simple:

Perform first the incubation with Goat Anti-Y, then wash, then followed by incubation with Donkey Anti-goat, then wash, then incubate Mouse Anti-X, then wash, then incubate with goat Anti-mouse! Ready!

You should ommit BSA when dealing with goat secondary antibodies.

However, simultaneously all primary antibodies together and later the secondary ab will not work!!!

Good luck!

Stefan

I would strongly recommend against trying to use primary and secondary antibodies raised in the same species in double labeling experiments like you propose. You are just begging for false positives!  Even if you image each channel seperately and your red channel is clean,  you will still have false-positive Alexa488 signal present in your green image. When you overlay the two images to assess the localization of the antigens, you will not be able to distinguish what double labeling is real and what is false.

The Donkey anti-goat Alexa596 secondary antibody will bind to BOTH your Goat anti-Y and your Goat anti-Mouse-Alexa488. This will give you some false positive labeling. Standard secondary antibodies have 2 antigen binding sites on them. When the Donkey anti-Goat Alexa596 binds to Goat anti-Y,  it will still have one of its binding sites available to bind to another Goat IgG.  This second unoccupied binding site will be available to bind to your Goat anti-mouse Alexa488.  This will create a complex of Donkey anti-Goat 596 PLUS Goat anti-mouse Alexa488 bound onto your Goat anti-Y primary, producing false-positive double labeling.

I can think of three potential solutions. Options 1 and 2 are the better choices:

1)   The best option would be to get an Anti-Y or an Anti-X primary antibody from a non-goat species for your experiment if such antibodies are available.

2)   Get an anti-mouse Alexa488 secondary antibody raised in some species other than goat (or sheep as most anti-goat secondary antibodies will happily bind to sheep Igs as well). There are many host options available these days-- rabbit, chicken, hamster, etc....  If you choose a secondary antibody raised in a rodent (rat, hamster, etc) you could experience cross-reactivity with the mouse Alexa488 secondary. Rabbit may be the best bet here.

3)    You might be able to dodge the unwanted binding between the Donkey anti-goat Alexa596 and the Goat anti-mouse Alexa488 antibodies problem by following the sequential labeling procedure suggested above by Stefan but substitute an Fab-fragment Donkey anti-goat Alexa596 secondary antibody for the standard Donkey anti-Goat Alexa596 antibody that you have. The Fab fragment has only one binding site on it, and once bound to the Goat anti-Y it would not be able to bind to the Goat anti-mouse Alexa488.

Hope this helps. Good luck.

Dave Sherry

I agree with David Sherry. You'd be begging for a false positive. There are plenty of different Donkey secondaries available.  Just change your goat secondary for a donkey anti-mouse and your assay should work.

Hello Together,

the way I described is feasible and works very well. We did own experiments by co-labelling Anti-BrdU and Anti-Tyrosine Hydroxylase, or Anti-BrdU and Anti-GFAP / Anti-IBA1  in brain tissues. This was done with such antibodies mentioned on top.

However, I agree it is better to use different and appropriate first and secondary AB-species. But sometimes it is not feasible. For example because you have to use a specific Primary antibody and in such combinations, perhaps,  secondary AB are only available from low amounts of species (species reactivity and conjugation is sometimes limited).

Sincerely Yours

Stefan

I know is possible, and I also know there are times when you don't have another possibility because of the lack of reagents. However, basing on the experimental layout described in the original post, the secondary he needs is a donkey anti-mouse-AF488 (certainly available in the market). As the reagent is easily founded I don't see any reason to try a protocol with potential for false positives instead of using the basic protocol.

If you are working with primary abs of different animales you can apply them simoultainisly  in a cocktail. You can do this as well with the secondary abs., but this makes it necessary to use either secondary abs  from the same host e.g. Horse-Anti-Mouse + Horse-Anti-Gooat or in your case use instaed of Goat-Anti-Mouse Horse-Anti-Mouse or Donkey-Anti-Mouse. Then you are absolutly  on the save side. In my opinion  all other options will lead partially to false positive colocalisation results.

if you can afford to give up a little signal, it is best to label the primary antibodies directly with the fluorophores. Different species antibodies are then not needed.  If not feasible at least reduce the risk  of cross reactivity by labeling one of the primary antibodies with biotin, and use a fluorophore labeled streptavidin in place of one of the secondary  antibodies.  Even then it would be prudent to confirm the specificity of each batch of the remaining secondary antibody.

I agree with Stefan, applying the antibodies simultaneously won't work.

The way around it is to perform individual incubation for each antibody and add a proper washing steps in between.

Best,

First of all will the antibodies work if raised in the same species? Secondly simultaneous application of both antibodies may not help you at all. Best way is using syncronous sections for each antibody separately and use image merging for representing results if required to be in one plate.