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
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.
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.
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
Histonet mailing list