Posted by
Gilbert Bigras on
URL: http://imagej.273.s1.nabble.com/DAB-quantification-tp5006159p5006169.html
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 [
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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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