Re: [Histonet] Holzer stain for glial fibers

From:"John A. Kiernan"

I don't think there are answers, other than wild
speculation, to your questions! A few statements
can be made about the reagents, but they don't
necessarily relate to Holzer's method.

When a PMA solution is applied to a section its
large anions stick to regions of protein that are
permeable and rich in basic amino acids. (This
happens in collagen, in trichrome methods.) PMA
also makes insoluble pigment precipitates when
mixed with cationic triphenylmethane dyes such as
crystal violet. Non-aqueous PMA may have tissue
affinity different from that of an aqueous
solution. With the chemically related
phosphotungstic acid, the solvent and other
factors influence the tissue components that take
up the metal when PTA is used as a contrast stain
for EM (see Hayat 1993 Stains and Cytochemical
Methods, various places in book).

The Holzer method I'm now reading (in H. Cook's
"Manual", 1974) does not have a reagent called
phosphomolybdic alcohol, but the PMA is dissolved
(0.5%) in 95% alcohol. 

I have no idea what the ethanol-chloroform mixture
does. As the solvent for the dye, it might help to
carry crystal violet into hydrophobic domains of
the section. Clearly care is taken not to have any
water in contact with the sections before, during
and immediately after the staining in non-aqueous
crystal violet.

Cook suggests that the KBr might act as a trapping
agen for previously bound dye. (This would be an
action similar to the iodine-KI solution used in
Gram staining.) 

The aniline-chloroform-ammonia mixture is used to
differentiate the stain. Ordinarily you wouldn't
expect a cationic dye like cresyl violet to be
extracted by an alkaline solution. Possibly it
extracts bound PMA, or dissolves a PMA-dye-bromide
complex out of hydrophobic but not out of
hydrophilic domains in the tissue. All guessing!
There's plenty of room for benchtop testing of
ideas with this one. It will be splendid if you
come up with hard evidence for a possible
mechanism for Holzer's stain.

Several years ago I was talking with a pathologist
who had tried to show that GFAP was the substrate
for Holzer's method. He failed to do so, using
methods equivalent to the ones he'd used to show
that Bodian's protargol method demonstrated one of
the neurofilament proteins. Nevertheless, it's
difficult to believe that Holzer's method and the
related Anderson's victoria blue staining anything
other than GFAP; is there another major strctural
protein in astrogliosis tissue?

Good luck with your research. I'd recommend
reading Holzer's 1921 paper; he must have
developed the method on the basis of some sort of
reasoning! I don't know of any published work on
the mechanism of Holzer's methods for gliosis.
Perhaps someone else will come up with something. 
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
_______________________________ wrote:
> Hello everyone,
>     I am doing research on the Holzer staining  technique, I was wondering if
> anyone could tell me the functions of the  following reagents: 0.5% Aqueous
> Phosphomolybdic Acid solution, Phosphomolybdic  Alcohol solution, Absolute
> Alcohol Chloroform mixture, and Potassium Bromide  solution.
> Thank you!

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