Posted by
Hugo A. M. Torres on
Feb 20, 2009; 2:05am
URL: http://imagej.273.s1.nabble.com/Re-Nuclear-volume-ploidy-and-Stain-stochiometry-tp3693629p3693631.html
On Wed, 2009-02-18 at 13:48 -0500, Alton D Floyd wrote:
> For autoradiographic grain counting, the type of emulsion, developer
> used, and method of development all influences the grain size and
> morphology. When this technique was widely used, in the early 1960's,
> most investigators used developers that produced tiny, round grains. As
> in any other type of quantitative measurement, the specimen preparation
> and grain development must be highly controlled.
>
> When I was actively involved in this type of work, we found that we had
> to create new calibrations each time we used a new batch of emulsion.
> The calibration was created by making a series of slides, coating with
> emulsion, and exposing for increasing lengths of time. Then we took
> reflectance measurements, and compared these to actual grain counts in
> the measured areas. With such a calibration, we found excellent
> correlation between actual counts and reflectance measurements.
> Obviously, the measurements were much faster, and permitted studies such
> as localization of radioactive tagged materials in serial sections of
> organs, such as rodent brains.
> I don't remember all of the citations to these type of studies. I will
> check my old citation card file and see if I can come up with specific
> citations that will give you a starting point for a citation search.
Excellent! I am interest in how those control experiments should be
conducted in more detail, I could start by reading one of your papers.
Would you recommend one in particular?
Setting aside the problem of silver particle size in emulsion
radiography for a moment and back to the problem of DAB
semi-quantification, Dr. Al Floyd said previously:
> ... DAB cannot be used for absorption photometry, as it does not meet the Beer-Lambert criteria for
> a phometric material. It is not a true absorbing material, as it is
> actually a particulate (the reason it is an effective stain for electron
> microscopy). As the concentration of DAB in a stained specimen
> increases, more and more light is scattered outside the capture cone of
> the objective, and therefore any measured signal is very nonlinear.
Dr Floyd, sir, after reading this statement I am really intrigued: an
article from the American Journal of Pathology (Open Access PMID:
11159179) claims that at least three times the linearity between DAB
absorbance or concentration staining and antigen concentration was
established by incorporating antigens and antibodies at different
concentration combinations in mounting media and therefore suitable for
densitometry (references number 32,34,43).
Unfortunately I don't have access to these works from my institution and
I can't ponder on their quality as of now.
I did find another work though, where a similar approach was taken and
it seemed pretty robust for me. Now, I am not particularly experienced,
so I would like if you could take a critical look (or anyone else in
this list, I gladly want to hear): PMID 8773563
Also mentioned:
> It might be assumed that one could create a calibration chart, much as
> was done with photographic grains for counting of autoradiographs.
> This does not work for DAB, as the particle size changes from
> manufacturer to manufacturer and with age of the DAB solution.
True, but by creating standards one would still be able to compare,
semi-quantitatively, relative amounts of antigen between experimental
and control groups, say, expression levels of neuropeptide P between
transgenic and naive mice groups.
--
Hugo Arruda de Moura Torres
==================================
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