stained or not (use a nissl counterstain to coun unlabeled cells). You can
use stereology to do this quantification in an unbiased way. For more info
bivariate relations in the nervous system under different conditions. J
Gundersen HJGJ, E.B. : The efficiency of systematic sampling in stereology
and its prediction. J. Microsc 1987
estimates: a computer simulation comparing three estimators. J Microscopy
> Hello Yiota
>
>
> There is indeed proportionality between the intensity ant the amount of
> antigen but the proportionality is unpredictable for the following reasons:
>
> 1) as Gabriel wrote, most stains are not stoichiometric and the main
> reason I believe is the more or less random (or not controlled)
> amplification methods used by detection kit used in IHC lab (polymer or
> multimer attached with multiple enzymes like Horseradish Peroxidase for
> DAB). So the proportion of one antigenic site and the number of one
> (chomogens) signals is unknown
>
>
> 2) If you deal with a stoichiometric stain, the relationship between the
> amount of antigen and the intensity is not linear... but logarithmic. (See
> Beer-Lambert law and related integrated optical density IOD)
>
> 3) finally DAB is the worst stain for quantification: it violates the
> Beer-Lambert law: in addition to absorb light like others stains, when
> abundantly present there are clots of the chomogen which diffract light
> which therefore provides a stronger intensity than explained by light
> absorption for a given amount of antigen or substrate.
>
>
> Best
>
>
> Gilbert Bigras, M.D., Ph.D., FRCPC (Path)
>
>
> Cross Cancer Institute
> 11560 University Avenue
> Edmonton, Alberta
> Canada T6G 1Z2
>
> Associate Clinical Professor
> Department of Laboratory Medicine & Pathology
> University of Alberta, Canada
>
>
>
> ________________________________
> From: Panayiota Ploutarchou <
[hidden email]>
> To:
[hidden email]
> Sent: Friday, January 17, 2014 9:24 AM
> Subject: Re: DAB quantification
>
>
> Hi Gabriel,
>
> Thanks for your answer. I agree that I can't quantify, but doesn't darker
> (and more) DAB staining correlate to more antigen present? I was just
> wondering whether there is some sort of semi-quantitative way to go about
> doing this...
>
> Many thanks for your help,
> Yiota
> ________________________________________
> From: ImageJ Interest Group [
[hidden email]] on behalf of Gabriel
> Landini [
[hidden email]]
> Sent: Friday, January 17, 2014 4:19 PM
> To:
[hidden email]
> Subject: Re: DAB quantification
>
> On Friday 17 Jan 2014 15:42:22 Panayiota Ploutarchou wrote:
> > So, I've done Immunohistochemistry on tissue sections, and finished with
> DAB
> > staining (which basically gives brown staining at the locations where a
> > reaction with the antibody took place). Now, I would like to quantify
> that,
> > but the pixel intensity function of Image J seems to give weird results
>
> Unfortunately IHC is not stoichiometric and so you won't get a reliable
> measure of how much product you have. Most stains are not "quantitative"
> (with
> some exceptions like Feulgen and phalloidin for example).
>
> > and also how would you suggest I go about quantifying
> > the DAB staining as I would need to compare staining in control and
> treated
> > sections.
>
> You can't, immunostains are not a quantitative technique. What you see as
> dark
> has no accurate relation to the amount of antigen present, which is what
> the
> purpose of the quantification is.
>
> Cheers
>
> Gabriel
>
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Michelle M. Naugle