Re: [Histonet] Timm silver sulphide method...
For insoluble metal-containing deposits, yes.
But the zinc in the brain is in solution
in certain synaptic vesicles. The zinc in
leukocytes and Paneth cells stays in place
after alcohol fixation and can be stained
with dithizone, which is a less sensitive
method than Timm's.
Greg Dobbin wrote:
> Hi Chris and John,
> Chris, you did not say which metals you hoped to stain.
> John you state:
> "Timm's method simply won't work (or not
> in any meaningful way) on formaldehyde-fixed
> paraffin-embedded specimens."
> While I do not have any experience with the method described by Chris using perfusion, I have on many occasions successfully stained FFPE sections with a modified Timm's Stain for heavy metals in general (ie not distinguishing one metal from another) and for copper specifically. I have offered the method and reference several times over the years here on the Histonet.
> The reference again is:
> “Diagnostic Histochemistry”; Frederick T. Zugibe
> Copyright 1970 by The C.V. Mosby Company
> (Standard Book Number [ISBN?] 8016-5702-4)
> If either of you wish to receive a copy of the method with a few of my own observations, I would be happy to send it along in the form of an attachment. Just indicate whether you want it in a WordPerfect or Microsoft Word format.
> Date sent: Thu, 04 Nov 2004 11:21:19 -0500
> From: John Kiernan
> To: Chris.Goodall@bristol.ac.uk
> Copies to: email@example.com
> Send reply to: firstname.lastname@example.org
> related fields
> Subject: Re: [Histonet] Timm silver sulphide method for light
> microscopiclocalisation of heavy metals.
> > 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.
> > ___________________________
> > Chris.Goodall@bristol.ac.uk 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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> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Greg Dobbin Research Technologist, Pathology Lab, Atlantic Veterinary College, U.P.E.I. 550 University Ave. Charlottetown, P.E.I. Canada, C1A 4P3 Phone: (902)566-0744 Fax: (902)566-0851 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Happiness is a journey, not a destination.
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