Gregg Dobbin's Timm's/Cu/Zn/Chemistry post

From:mike king <> (by way of histonet)

Greg Dobbin <dobbin@Upei.CA> wrote:
Subject: Timm's/Cu/Zn/Chemistry
...My first problem:
	I want to stain for copper with the Danscher Modification of
Timm's stain for heavy metals *and*, (here's the rub) stain for
metallothionein by IHC on the same sections....Question #1:	Is there a way
to quench endogenous peroxidases without oxidizing?

Sodium azide will inhibit peroxidases but I don't know what effect it will
have on other metallic ions in the tissue.  It IS highly reactive with
certain metals and can be lethally toxic so know what you're doing before
you mess with it.  We use it at .002% in PBS to retard bacterial growth
during prolonged section incubations (e.g. primary, secondary antibodies),
but must not use it with any subsequent steps involving peroxidase
(avidinylated HRP incubation or chromogen reactions).  Is your endogenous
peroxidase problem so bad you have to quench?  Maybe you can just bypass it
altogether, or use a different visualization agent (fluorescence, alkaline

>My second problem:
	Does anyone know of a method to selectively demonstrate
zinc in formalin-fixed, paraffin-embedded tissues. I saw something
called Zincuin or Zinquin mentioned in an article but no details. Any

Zincon is probably the chemical to which you refer; it's available from
Sigma and described with references in the Sigma-Aldrich Handbook of
Stains, Dyes, and Indicators.  I've tried it on rat brain tissue after
either sulfide/formaldehyde (Timm's) perfusion or conventional formaldehyde
fixation but did not see anything like the metal sulfide/silver Timm's
histology.  It is probably not sufficiently sensitive to demonstrate the
small amounts of zinc present in the tissue.  Also, unless the zinc is
converted to insoluble sulfides in tissue (during perfusion/fixation) most
of it will diffuse out during processing.  So unless you prepared to do
Timm's from the beginning you aren't likely to have much luck detecting
zinc in your material because it just won't be there.

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