Re: HEMATOXYLIN ORDER
|From:||"J. A. Kiernan" <firstname.lastname@example.org>|
On Sun, 15 Oct 2000, Lee & Peggy Wenk wrote:
> Does it matter what order the reagents are added together to
> make an aluminum salt-sodium iodate hematoxylin, such as
No. The iodate oxidizes the haematoxylin to haematein,
which then forms a dye-metal complex with aluminium ions. The
complex (with excess aluminium) is the staining reagent.
Aluminium salts do not interfere with the oxidation of
haematoxylin to haematein, so you can just chuck everything
in and leave it on a magnetic stirrer until everything has
I like to dissolve the haematoxylin and iodate first (because you
can see that they have dissolved) and put the alum in last, but
this isn't necessary. For an air-ripened mixture such as Ehrlich's,
the alum and other ingredients all go in long before any of the
haematoxylin becomes oxidized to haematein.
For a long life, only part of the haematoxylin should be converted
to haematein by iodate (instantaneous) oxidation. This leaves a
reservoir of haematoxylin that is air-oxidized to haematein over
the course of several weeks, to replace the haematein lost by
further oxidation (to useless products) and also that which is
removed from the solution by uptake into stained sections.
These stains should really have names like "Harris's haemalum," not
"Harris's haematoxylin" because haematoxylin does not contribute to
the imparted coloration. It is the precursor of one component of the
haematein-aluminium complex. The idea of adding only enough iodate
to oxidize half the haematoxylin (thus prolonging shelf life) is
quite modern (about 1950 I think). The early authors prescribed
enough oxidant to change all the haematoxylin into haematein,
except in the case of the slowly ripening air-oxidized formulations,
which still have the best keeping properties. The name "haemalum"
has been around for many years, but doesn't seem to be much used
in North America. It nicely takes care of all solutions that contain
haematein or a mixture of this dye with haematoxylin.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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