Detection and amplification in immunohistochem
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | Histonet <Histonet@pathology.swmed.edu> |
Reply-To: | |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
This is a general answer to an enquirer who sent in an
impossibly long wish list.
It's important to appreciate that when amplifying methods are used
(PAP, ABC, tyramides etc) the intensity of the staining (whether
fluorescence or ordinary colour) does not increase with the local
concentration of the antigen. Simple immunofluorescent ("sandwich")
methods are more likely to provide significant simple comparisons
of the "brighter = more antigen" type, but only in quite a crude
way. Unless you test many different concentrations of all the
immunoreagents and detection systems, immunohistochemistry can only
say either "Present" or "Absent or not enough to detect." High local
concentration of an antigen in tissue can make immunostaining weaker,
and this fact (fully substantiated in the peer-reviewed literature
for 20 years) is often ignored.
There are lots of textbooks and review articles about all this.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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