Posted by
Hugo A. M. Torres on
Apr 08, 2009; 3:01pm
URL: http://imagej.273.s1.nabble.com/regarding-IHC-PROBLEM-tp3693036p3693040.html
Dear Santosh,
As G. Landini and Al Floyd mentioned, DAB is not in rigor a truly
absorptive material and you might have trouble publishing your data if
your method uses densitometry quantification based on the law of
Lambert-Beer.
It is very common, not withstanding, to find in situ hybridization
quantification of brains slices dipped into autoradiographic emulsions.
Silver granules, which likewise, do not satisfy Lambert-Beer criteria,
is quantified by densitometry in darkfield microscopy where light is
reflected!(A quick pubmed search will reveal)
Now, I don't want to justify an error with another one, the question is
whether there are more white pixels created by reflective silver spots
where there are molecules of interest. The rule that DAB can not be used
at all might not be a general rule and be more like a rule of thumb as
this can vary from antibody to antibody according to Watannabe et al(See
below). In case positive, although one can not estimate the true number
of molecules in situ on the basis of Lambert-Beer law, one would in
principle be able to compare rough relative amounts (e.g. a
semi-quantitative estimate of the proportion of molecules) in this
tissue site as opposed to another tissue site subjected to the same
conditions by photometric densitometry (granted exposure to the same
solutions, for the same amount of time, same illumination settings, lab
temperature, etc).
A couple of works have addressed this question. In the case of DAB
deposited by a antibody-biotin, avidin-peroxidase mesh, I recommend you
read this work:
HUANG, X.; CHEN, S.; TIETZ, E. I. Immunocytochemical detection of
regional
protein changes in rat brain sections using computer-assisted image
analysis. The
Journal of Histochemistry and Cytochemistry: Official Journal of the
Histochemistry Society,
v. 44, n. 9, p. 981–7, set. 1996. ISSN 0022-1554. PMID: 8773563.
And this
WATANABE, J.; ASAKA, Y.; KANAMURA, S. Relationship between
immunostaining intensity and antigen content in sections. The Journal of
Histochemistry
and Cytochemistry: Official Journal of the Histochemistry Society, v.
44, n. 12, p. 1451–8,
dez. 1996. ISSN 0022-1554. PMID: 8985137.
For a more in-depth description of the main sources of variation in
Imunohistochemistry I recommend this:
TAYLOR, C. R.; LEVENSON, R. M. Quantification of
immunohistochemistry–
issues concerning methods, utility and semiquantitative assessment II.
Histopathology,
v. 49, n. 4, p. 411–24, out. 2006. ISSN 0309-0167. PMID: 16978205.
And finally, to avoid uninformed mistakes:
ROSSNER, M.; YAMADA, K. M. What’s in a picture? the temptation of image
manipulation. The Journal of Cell Biology, v. 166, n. 1, p. 11–5, jul.
2004. ISSN 0021-9525.
PMID: 15240566.
Hugo Torres
On Tue, 2009-04-07 at 20:09 +0530, Santosh M wrote:
> Thank you for your reply
> according to mailing list archive: DAB cannot be used for absorption
> photometry, as it does not meet the Beer-Lambert criteria for a
> phometric material.
> (
http://n2.nabble.com/Re--Nuclear-volume,-ploidy-and-Stain-stochiometry-td2347676.html)
> But I feel Color Deconvolution plugin it is at least differentiating colours.
> I have seen some articles in which they have used for quantitative
> immunohistochemistry for DAB stained slides softwares like Image pro
> plus and Q-IHC.
>
> So What is the best solution available for quantitative
> immunohistochemistry for DAB stained slides?
>
> Thanks for any suggestions
>
> Santosh M
>
>
>
> 2009/4/7 Gabriel Landini <
[hidden email]>:
> >> Santosh M wrote:
> >> > How can i quantity only leaving out Haematoxycillin ?
> >> > its urgent plz
> >
> > On Tuesday 07 April 2009 11:37:41 Przemko wrote:
> >> Use Color Deconvolution plugin. IT IS GREAT!!!!!!!!!!!!!
> >
> > You can separate the stains that way, but I you must be aware that DAB
> > staining cannot be quantified reliably by its intensity.
> > This has been raised several times here already. I would suggest to search the
> > mailing list archive to learn why.
> >
> > Cheers,
> >
> > G.
> >