Hi every body,

I am using a Bio-Rad's MRC 600 system to localize the PKC isozymes in
preimplantation stages of mouse embryo and I am interested to
quantitate the expression of these isozymes (or better to say that I
would like to compare the expression levels of these isozymes between
different preimplantation stages). I would be grateful if you read
the following part which shows the way I am going to do this and
correct me where you feel there is a problem:

The size of the embryo is 100 micron in these stages, however, the
number of cells increases from one stage to the next (e.g. 2-cell,
4-cell,
8-cell,...). The 5 micron image stacks will be recorded with the same
adjustments and without any processing for each embryo exposed
to an isozyme-specific antibody and then these stacks will be
reconstructed using Bio-Rad's CoMos software.  The reconstructed
file will be converted to TIFF format using Confocal Assistant software
(version 4.02). And, to compare the expression of PKC isozymes
between embryos at different developmental stages, the mean pixel
value and standard deviation of fluorescence intensity will be
determined
for each embryo exposed to individual antibodies against each isozyme
using Scion Image software. Since embryos with different number of cells

may show different fluorescence intensity, the mean  pixel value will be

divided by the number of cells to acquire the mean of cellular
fluorescence.

I guess the main question is that" is the mean pixel value of a
reconstructed
image equal to sum of the mean pixel values of constituting image
stack?".


Thank you for your help.


Hesam Dehghani, DVM, PhD candidate
Department of Biomedical Sciences
University of Guelph
Guelph, Ontario