Louise,

It's been a while since I've done semi-quant analysis of IHC sections,
and you are right to be concerned about such issues. Section thickness
will impact staining intensity.

We used a similar integer scale of 0 to +4 to indicate chromogen
intensity within a given experiment. Generally, it was sufficient though
admittedly imprecise. Such are the limitations of quantitative (even if
semi-) observations in IHC... or ISH for that matter.

Consider also variable levels of intracellular expression and the plane
of the section within particular cells. If you have a perinuclear
antigen, for example, and one cell with exposed surface (from cutting)
with nuclear material is evident, then that cell will "stain" more
intensely than a neighboring cell which might have quantitatively more
antigen but has not been cleaved near the nucleus, but which may
actually appear to be a measurably larger cell due to the nucleus being
localized to one pole.

Without getting too exact about an inexact observation technique, the
checks we implemented were to note the relative counterstain intensity
relative to the IHC chromogen within sections of a given experiment, to
not extrapolate observations from one species to the next or from one
tissue to the next, and to get several observers corroborate any
"measurable" observations. To elaborate on the first point, if the
counterstain intensity in one section is measurably higher than another,
complementary section, then that usually indicated to us that the
section was thicker; consequently, that comparison was discarded. Also,
apart from having multiple observers quantify the final product, it
helps to have terrific and highly experienced staff to create the source
palette. And lots of previous experience observing the tissue/species of
interest. I'm not sure if that is really quantifiable either. ;)

Good luck with your study, and by all means, review Rene J Buesa's paper
and others like it. Semi-quant-IHC a tough nut to crack.

Todd Sherman

HistoSoft Corporation
"Biology in a new form..."
Home: www.histosoft.com
Member Services: www.myhistosoft.com


histonet-request@lists.utsouthwestern.edu wrote:
> Today's Topics:
>    9. IHC staining intensity. (louise renton)
> ----------------------------------------------------------------------
> 
> Message: 9
> Date: Wed, 30 Nov 2005 12:13:17 +0200
> From: louise renton 
> Subject: [Histonet] IHC staining intensity.
> To: Histonet@lists.utsouthwestern.edu
> Message-ID:
> 	
> Content-Type: text/plain; charset=ISO-8859-1
> 
> Dear all,
> 
> I am currently in discussion with someone doing research who is using
> a semi quantitative method of reporting stainig using INTENSITY of
> cytoplasmic staining as a means of quantifying results.  Staining for
> a specific antibody is reported as ranging from zero to 3+.  What I
> would like to know is whether IHC intensity is influenced by the
> thickness of the section. What I mean is, if staining is seen in a
> whole cell, or one that has been bisected - will there not be a
> variation in that one will appear darker when compared to the other?
> Or is this variation minimal? Is my logic fuzzy?
> 
> I appreciate any input on this matter
> 
> best regards
> 
> 
> Louise Renton
> Bone Research Unit
> University of the Witwatersrand
> Johannesburg
> South Africa
> "....onwards through the fog!"
> 
> ------------------------------


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