[Histonet] Re: Two questions about double IHC

From:"Johnson, Teri"

Scott wrote:

I think there might be a way to do this.  The tyramide doesn't actually
bind to the immune complex but rather precipitates and covalently binds
to the tyrosine residues in the tissue proteins.  Therefore, I think you
could use the non-biotin fluorescyl-tyramide kit to stain your first
antibody, then elute the immune complex leaving the fluorescyl-tyramide
behind.  Then you'd have to get a little tricky.  Since both of your
antibodies are rat anti-mouse, you'll have to pre-label your other
primary somehow.  The easiest thing to do would be to biotinylate that
primary.  Alternatively, you could pre-complex your biotinylated
secondary Ab with your second primary and block the unbound secondary Ab
with mouse serum so that it doesn't stick to your first primary
antibody.  You can figure out the concentrations that work by yourself
or you could use the biotinylation and blocking reagents from the Dako
ARK.  After you've done that, you can put the primary/secondary complex
on the tissue and follow with your biotin-based TSA and a
Streptavidin-Texas red.

Anyway, that's what I could come up with.  I know it sounds really
complicated but I think it might work (with a LOT of trial and error to
get it right).  Does anyone out there have any input on this?

Scott Turner
Schering-Plough Biopharma
(formerly DNAX Research Institute)
Palo Alto, CA

Actually that does sound doable although still complicated. I'm always
concerned about residual molecules of the first binding with the
detection of the second, or losing some of the signal of the first after
elution. It would take a lot of work to make sure what you're seeing is
real. Nice job, Scott.

If you can get this simplified, using one biotinylated primary antibody
and separate detections systems, I think that's the best. 

I think Albert Einstein had it right when he said: "Make everything as
simple as possible, but not simpler."

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

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