Re: does the spectrum of dab change with the addition of blue hematoxylin
As I haven't seen an answer posted I'll give you my $0.02 worth. As
this is not a trivial question when it comes to image analysis.
No there is not (that I am aware of) an actual chemical reaction between
the DAB and Hematoxylin. It would seem to answer the question but it is
not quite that simple.
The normal use for DAB is to demonstrate the sites of antibody binding
to a specific epitope and the normal use for Hematoxylin in conjunction
with DAB immunohistochemistry (IHC) is as a nuclear counter stain which
allows us to visualize the areas of interest (DAB pigment) in relation
to the morphology of the tissue section as a whole. If the antigenic
sites of interest are located in the nucleus of the cell the DAB
reaction will be located there but only where the epitopes are. This
is because the IHC reaction is very specific (when it's done right and
the Moon is in the right phase). Hematoxylin by comparison is not that
specific. It is a basic dye which binds to the acid components of the
nucleus (not the best explanation but it will suffice for now) and
other cellular structures. Therefore if the epitope is located on a
place that the hematoxylin would normally stain then there will be some
hematoxylin staining there also. This is why most of the hematoxylins
used as counterstains for IHC are either diluted or do not stain as
intensely (like Mayer's) as Harris's and it's variations. This is why
some tissues on which IHC image analysis (IA) is to be performed will
not be counterstained or a different dye will be used instead of
hematoxylin as the intensity of the DAB reaction varies with the amount
of epitope present among other considerations.
I'll not go into how this doesn't effect some IA systems as I've rambled
on long enough.
The answer is 1- yes 2- no
Andrew Rabinovich wrote:
>I am a computer scientist and thus this question may seem trivial. Does
>the spectrum of dab change as the blue hematoxylin stain is added to the
>sample. In other words, is there a chemical reaction between dab and
>hematoxylin once both are applied to the tissue.
>Thanks a lot,
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