Re: [Histonet] Timms copper stain

From:John Kiernan

Your recipe for the developer does not include a protective colloid, so the mixture will be unstable. 
Try the developer of Danscher & Zimmer (1978) Histochemistry 55: 27-40.
The first three ingredients of the developer may be mixed several hours in advance, but the last (AgNO3) is added immediately before using.
50% (w/v) aqueous gum arabic stock solution: 60 ml; Citrate buffer: 10 ml; Hydroquinone (5.67% w/v, aqueous): 30 ml.  Silver nitrate (17% w/v, aqueous stock solution): 0.5 ml.  The buffer is: Citric acid (monohydrate) 25.5g, Sodium citrate (dihydrate) 23.5g, dissolved in water and made up to 100 ml. The pH should be 3.7 if an adjustment is necessary use citric acid or diulute NaOH; not hydrochloric acid, which would precipitate the silver out of the developer.
Remember that Timm's and related sulphide-silver methods are not specific for copper. They detect any metal with an insoluble sulphide, including iron, zinc and many unphysiological metals that might be present in tissues, such as mercury or lead. If an alcoholic fixative is use (eg hydrogen sulphide dissolved in ethanol) these methods will also detect calcium, because CaS is insoluble in alcohol. 
There are histochemical methods for copper that are quite specific, though less sensitive than Timm's technique. The specific methods detect abnormal deposits of Cu (as in wilson's disease) but not the copper in normal mammalian tissues. Unless your kidney tissue contains much more copper than zinc, the silver deposits that you see in the sections will not mean anything. For more information see the Danscher & Zimmer paper cited above; also more recent publications by Danscher. The method can be tweaked to make it fairly specific for zinc or mercury. I don't think this can be done for copper, but could be mistaken.
John Kiernan
Anatomy, UWO
London, Canada
----- Original Message -----
From: Sarah Glyn-Jones 
Date: Monday, October 29, 2007 23:39
Subject: [Histonet] Timms copper stain

> Hi,
> I have recently begun trying to optimise Timms copper stain on kidney
> tissue. I have frozen tissue sections and I leave them in 1% 
> sodium sulphide
> for 1hr before TCA treatment and then the Timms reagent.
> The recipe I am using is:
> Solution A
> 5% silver nitrate aqueous (5g in 100ml distilled H2O).
> Solution B 
> Hydroquinone                          2.0gms
> Citric acid 
> (monohydrate)          5.0gms
> Distilled 
> water                         100ml
> Develop sections in freshly filtered solution of 1 part A and 5 
> parts B for
> 5min
> There are a number of different recipes for the Timms reagent 
> and the one I
> am currently using is not working. I cannot seem to get the 
> stain into the
> tissue. I end up with lots of staining on top of the tissue 
> despite heavy
> washing. 
> Does anyone have a different recipe that I could try? I have 
> seen recipes
> for Timms reagent using gum arabic and 10% hydroquinone, except 
> hydroquinoneis only soluble in water to 7% (I tried making a 10% 
> solution and it was a
> disaster).
> Any suggestions would be greatly appreciated.
> Thanks!
> Sarah
> PhD Candidate
> Proteomics and Biomedicine Group
> School of Biological Sciences
> Thomas Building
> University of Auckland
> Tel: (09) 373-7599 ext 85763
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