[Histonet] Double stain IHC question

[Caroline Miller]

+ to Gudrun's comments.

My addition is that fluorescence labels may work for this application.
Again, depending on the species of the antibodies. The other advantage of
the fluorescence is that you would be able to truly see the colocalization
(yellow for instance if you used red and green fluorescent antibodies).
Rather than the muddiness I have often got when trying two antibodies in
bright field chromagenic stains.

Remember though autofluorescence is greatly increased with paraffin
tissues, that may put another issue on your plate.

Good luck!

yours,
mills

On Sat, Feb 20, 2016 at 3:47 AM, Gudrun Lang via Histonet <
histonet at lists.utsouthwestern.edu> wrote:

> In my opinion, this would only be possible, if the commercial and the
> homegrown antibody are from different species. For example one from mouse
> and one from rabbit.
> Then you can proceed with different secondaries (goat anti mouse conjugated
> with peroxidase, goat anti rabbit conjugated with alkaline phosphatase).
> Then chromogens that work with each of the enzymes.
>
> If the antibodies are from the same species I see no way to distinguish
> both. Only if one is conjugated with biotin and the other with digoxigenin,
> then you could proceed with secondaries against biotin and digoxigenin.
> etc..
>
> Gudrun
>
> -----Ursprüngliche Nachricht-----
> Von: Judi Ford via Histonet [mailto:histonet at lists.utsouthwestern.edu]
> Gesendet: Samstag, 20. Februar 2016 01:54
> An: histonet at lists.utsouthwestern.edu
> Betreff: [Histonet] Double stain IHC question
>
> Hi everyone,
> I have a question in chromogenic double staining. Here is the situation.
>                 Tissue = human, frozen
>                 Antibody = same protein (A)
>
> 1.       Commercial antibody of A
>
> 2.       Homegrown antibody of A, human, biotinylated
>
> Question: can you stain both versions of this antibody on the same tissue,
> same slide? Goal is to see where each stains in the tissue and if they
> co-localize. If they do co-localize then how do you distinguish between
> that
> and where they stain individually? Would you use different chromogens and
> hope that where they come together it turns a different color?
> I am really interested if this can work. Thanks in advance for any replies.
> Judi
> South San Francisco, CA
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-- 
Caroline Miller (mills)
Director of Histology
3Scan.com
415 2187297