Re: does the spectrum of dab change with the addition of blue hematoxylin

From:Robert Schoonhoven

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

Robert Schoonhoven

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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