[Histonet] fluorescent peroxidase substrate questions


Look up tyramine/tyramide, the basis for the signal amplification (TSA) 
for peroxidase.  I think it is fluorescent and a great substrate, or you 
could use biotin-tyramide to pile biotins at the site, then use 
avidinylated fluors.

As for quantifying expression, you'd have to figure out a way to 
generate concentration standards and react them like your sections, then 
you _might_ be able to convince skeptics that your image analysis values 
were meaningful.  It ain't trivial though.

--Mike King
Date: Tue, 26 Jun 2007 15:37:04 -0400
From: "Turner, Scott" 
Subject: RE: [Histonet] HRP Substrates

While we're on the subject of HRP substrates, does anyone know of an HRP
substrate that will fluoresce?  I know that some Alk-Phos substrates
like Vector's AP Red do, but I'm looking for an HRP that will work for a
particular IF assay we're working on.

Scott Turner
Schering-Plough Biopharma
Palo Alto, CA

My questions are:  Assuming that all the parameters were kept 
equivalent within a "batch" of slides:
Can it be used in image analysis to quantify expression of a particular
epitope of interest among different samples in a batch of stained slides?
Or is DAB precipitate more of a random quantity depending on substrate
availability/enzyme activity and therefore cannot be used to compare 
among  slides (I realize that enzyme kinetics make the word "random" a 
bit of  an contradiction in this sense)?  This is based more upon the 
premise  that the fluorescent secondary only has so many places to bind 
vs. HRP that  creates more color the longer it works......(at least 
until the reaction is  stopped or the substrate/cofactors are exhausted).


Albert C.  Grobe, PhD
International Heart Institute of Montana Foundation
Tissue  Engineering Lab, Saint Patrick  Hospital

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