Re: [Histonet] Timm silver sulphide method for light microscopiclocalisation of heavy metals.

From:John Kiernan

The perfusion of sodium sulphide is necessary
to precipitate metal ions (principally zinc),
which are otherwise soluble. Relying on autolysis
to generate sulphide and precipitate the zinc
doesn't make sense because the zinc ions would
have diffused away from their original synaptic
sites, and the tissue would be decomposing
anyway. Timm's method simply won't work (or not
in any meaningful way) on formaldehyde-fixed
paraffin-embedded specimens. 

Glutaraldehyde is not the only permissible
fixative after perfusing the buffered Na2S.
An alternative is to use an alcoholic fixative
that has been saturated with H2S gas. It's
unlikely that your health & safety regulations
will allow this if they won't let you take a
little jar of glutaraldehyde into the PM room!

The best account of modern Timm staining is 
probably Danscher,G & Zimmer,J (1978)
Histochemistry 55: 27-40. Danscher has also
published many more recent papers on 
histochemical detection of zinc, mercury and
other metals with sulphide-silver methods.

John Kiernan
London, Canada.
___________________________ wrote:
> Dear All,
>     I have been asked to try the Timm method on some FFPE samples of
> human brain. The papers I have read give methods for perfusing rat
> brain with sodium sulphide solution followed by 3% gluteraldehyde in
> 0.15M phosphate buffer, and further treatment with sodium sulphide
> solution, or, post mortem brain samples snap frozen and frozen
> sectioned. However health and safety rules here do not permit frozen
> sectioning of unfixed human brain, or the use of gluteradehyde in the
> embalming room, so I am going to attempt this method on formalin fixed
> samples.My question is, has anyone experience of this method or any
> suggestions? It is mentioned that autopsy material left in situ for one
> or two days post mortem with no fixation may have enough endogenous
> sulphide ions or sulphide groups in the tissue due to autolytic
> activity and sulphide treatment may not be necessary, or if it is
> necessary, does anyone have experience of timing in the sulphide
> solution and is post fixation necessary after the initial formalin
> fixation. The paper also mentions that to prevent loss of
> metallosulphides present in the tissue the temperature of the wax
> should not exceed 50C which means the use of the dreaded low
> temperature wax, has anyone used conventional paraffin wax and got away
> with it?
> Sorry for the long request,and thank you,
> Chris Goodall
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