[Histonet] Double stain IHC question

[koellingr at comcast.net]

All, 
I guess I'm reading (or misreading) the situation differently than some.  That this is frozen, and not FFPE, is given originally. 
  
But I'm reading this as two different antibodies marking the very same protein. [Antibody=same protein A].  Is the original question can you mark same protein with two different antibodies to it?  I have done this with two antibodies to DIFFERENT epitopes of the same protein from home grown antibodies and we knew which antibody was directed to which epitope.  If two different antibodies are directed to the SAME epitope on the same protein they will just compete and favor that one with greater affinity/avidity/more conducive physical characteristics for binding.  Then colocalizing with fluorochromes is certainly possible as is done routinely. 
  
Ray (now in Spokane WA) 

----- Original Message -----

From: "Caroline Miller via Histonet" <histonet at lists.utsouthwestern.edu> 
To: "gu lang" <gu.lang at gmx.at> 
Cc: "Histonet at Lists. Edu" <histonet at lists.utsouthwestern.edu> 
Sent: Saturday, February 20, 2016 8:35:18 AM 
Subject: Re: [Histonet] Double stain IHC question 

+ 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 
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