regarding IHC PROBLEM

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regarding IHC PROBLEM

Santosh M
I am analyzing of imgae j software
but when i will try to adjust thershold level haematoxycillin stain is
interfereing with DAB
(following according to method mentioned on
http://rsb.info.nih.gov/ij/docs/examples/stained-sections/index.html
     i.e. Quantifying Stained Liver Tissue)
How can i quantity only leaving out Haematoxycillin ?
its urgent plz

regards

santosh
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Re: regarding IHC PROBLEM

Przemko
Santosh M wrote:

> I am analyzing of imgae j software
> but when i will try to adjust thershold level haematoxycillin stain is
> interfereing with DAB
> (following according to method mentioned on
> http://rsb.info.nih.gov/ij/docs/examples/stained-sections/index.html
>      i.e. Quantifying Stained Liver Tissue)
> How can i quantity only leaving out Haematoxycillin ?
> its urgent plz
>
> regards
>
> santosh
>
>  
Use Color Deconvolution plugin. IT IS GREAT!!!!!!!!!!!!!
Przemko

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LSD & Joints
O & N1, box 813- dark green
University of Leuven
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3000 Leuven, Belgium

phone: (32-16)34-61-96
fax  : (32-16)34-62-00
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Re: regarding IHC PROBLEM

Gabriel Landini
> 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.
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Re: regarding IHC PROBLEM

Santosh M
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.
>
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Re: regarding IHC PROBLEM

Gabriel Landini
On Tuesday 07 April 2009 15:39:34 Santosh M wrote:
> So What is the best solution available for quantitative
> immunohistochemistry for DAB stained slides?

Unfortunately there is no solution available.
Degree of DAB darkness is not indicative of expression of the antigen one is
detecting, which is why people do the 'quantification'.

Yes, some papers might have gone through the reviewing process, but you do not
know how many have been rejected by knowledgeable reviewers.

Cheers

G
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Re: regarding IHC PROBLEM

Hugo A. M. Torres
In reply to this post by Santosh M
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.
> >
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Re: regarding IHC PROBLEM

Santosh M
Dear Hugo Torres and G. Landini,

Thank you for your extensive information.

regards

Santosh

2009/4/8 Hugo A. M. Torres <[hidden email]>:

> 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.
>> >
>