Re: zinc detection (hippocampus)

From:"J. A. Kiernan"

dtata wrote:
> I forgot to mention in my original message that the reason we want to
> detect zinc is to estimate the volume of the mossy fiber system in the
> hippocampus. I am not very familiar with SEM techniques, but ...
> > X-ray microanalysis would be the easiest answer. If you have an SEM
> > with X-ray microanalysis and a backscatter detector system, you could

Zinc in the hippocampal mossy fibre system is nearly
always detected by Timm's sulphide-silver method or
one of its variants. The classic paper in this field
is Haug (1973) Heavy metals in the brain ... Adv. Anat.
Embryol. Cell Biol. 47:1-71. Timm's technique has been
improved over the years, especially in many publications
by Danscher, who was still publishing in this field in
the late 1990s and may still be at it.

The principle of Timm's sulphide-silver method is quite
simple. Fix the tissue as quickly as possible with a
fixative that contains sulphide ions, to precipitate
insoluble metal sulphides. Make sections and detect
insoluble metal sulphides by catalytic amplification
of the reduction of silver ions to silver atoms. 

This amplification step is similar to the development 
of a black & white photo, and it's still commonly called
"physical development," following one Liesegang who
coined the term in (?)1911. Physical development is
very much a chemical reaction! It is explained in 
textbooks of both photographic chemistry and 
histochemistry. This form of catalytic amplification
has also been used for many years by biochemists to
detect small amounts of protein in electropherograms.
It also occurs (subtly disguised) in all the silver
staining methods that selectively emphasize such
structures as axons and reticular fibres.

If you didn't fix your hippocampi in something that
precipitates zinc ions, don't expect to detect the
zinc in your sections, by EM or LM. There are plenty
of histochemical methods for detecting Zn ions, but
they will not work if the Zn ions are extracted from
the tissue during fixation, processing or the cutting
and mounting of sections. Most ordinary salts of Zn
are water-soluble. That probably includes all dissolved
zinc salts and many protein-bound zincs.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1


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