Posted by
Eric Scarfone on
URL: http://confocal-microscopy-list.275.s1.nabble.com/intensity-measurements-tp3005594p3008264.html
Hi,
I'll follow Julio's comment n°2 by adding that DAB staining is a
precipitate of the reaction product of peroxidase action on DAB in the
presence of H2O2.
SO you have to be carefull that it doesnt not reflect acuratelly the
number of antibody binding sites but rather it reflects the
concentration of DAB and its access to the preoxidase molecule (which
can vary a lot if your antigen is not on the surface).
Also the reaction is very dependant on the activity of H2O2 which in
turn is affected by light, age and god knows what.
So it is much more tricky to achiave a staining in a quantitatively
reproducible manner than Fluorescence (which is already not that
straight forward!)
Good luck
Eric
Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet
Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden
Work: +46 (0)8-517 79343,
Cell: +46 (0)70 888 2352
Fax: +46 (0)8-301876
email:
[hidden email]
http://www.ki.se/cfh/----- Original Message -----
From: Julio Vazquez <
[hidden email]>
Date: Monday, June 1, 2009 6:29 pm
Subject: Re: intensity measurements
To:
[hidden email]
>
> I should add a couple of things:
>
> 1. several of the manufacturers who sell digital pathology systems
> and/or image analysis software now have tools to separate and
> analyze
> color images such as those obtained from IHC chromogenic stains.
> Some may actually do a pretty good job at separating specific color
>
> ranges or hues. The problem here is that often the analysis has to
> be
> limited to measuring the stained area fraction, since in a color
> image, the "intensity" is a somewhat meaningless notion (you get
> the
> average of three values, such as red, green, and blue), and very
> different colors or hues may give similar "intensity" values.
> That's
> why separating color channels first and picking the one that's most
>
> representative is useful. In many cases though, measuring the area
> fraction that has the desired color range may be just fine. It all
> depends on the information that is most relevant for your experiment.
>
> 2. You also need to ask yourself whether DAB and other chromogenic
>
> stains are quantitative. You can generally distinguish pale from
> medium from dark staining, but the "dynamic range" is most probably
>
> more limited than, for instance, fluorescent staining. Doing some
> staining intensity calibrations would certainly be a good idea, to
> see how linear and/or quantitative this approach is.
>
>
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA
>
>
http://www.fhcrc.org>
> ==
>
>
> On Jun 1, 2009, at 3:51 AM, vaishali kailaje wrote:
>
> > hello friends,
> > We are interested in measuring the intensity in
> immnohistochemical
> > slides (DAB stained).
> > Could anybody suggest some software or a method to quantitate DAB
>
> > intensity in tissue sections.
> > Secondly,can these measurements be made using metomorph, ImageJ
> or
> > Axiovision as these are the softwares available with us.
> >
> > Thanks
> >
> > Vaishali Kailaje
> > Scientific Assistant,
> > Tata Memorial Centre,
> > Advanced Centre for Treatment Reasearch Education in Cancer
> > Kahrghar,
> > Navi Mumbai,
> > India.
> >
> >
> >
> >
> >
>
>