http://imagej.273.s1.nabble.com/regarding-IHC-PROBLEM-tp3693036p3693041.html
Dear Hugo Torres and G. Landini,
2009/4/8 Hugo A. M. Torres <
> 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.
>> >
>