Detection and amplification in immunohistochem

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From:"J. A. Kiernan" <>
To:Histonet <>
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 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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