Greg wrote:  "The quench [for endogenous peroxidase] is oxidizing the copper
and rendering it undetectable....Is there a way to quench endogenous
peroxidases without oxidizing?"

Why not try an IHC system that does not depend on a peroxidase substrate?
Then the problem of quenching endogenous peroxidase is eliminated
altogether.

Tim Morken, BA, EMT(MSA), HTL(ASCP)
Infectious Disease Pathology Activity
Centers for Disease Control and Prevention
Ms-G32
1600 Clifton Road
Atlanta, GA 30333
USA

PH: 404-639-3964
FAX: 404-639-3043

email: tim9@cdc.gov



-----Original Message-----
From: Greg Dobbin [mailto:dobbin@Upei.CA]
Sent: Thursday, September 07, 2000 10:44 AM
To: Histonet@pathology.swmed.edu
Subject: Timm's/Cu/Zn/Chemistry


Hello Histonet!

I hope my subject line attracted all the pertinent experts!
My first problem has to do with Timm's stain and copper, and the
second with detecting Zinc. I threw "chemistry" in the subject line to
tweek the chemists' interest!

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. I tried the Timm's
stain first, followed by the IHC, but the quench removed the Timm's
reaction product (reduced Ag). So, the next time I brought the
sections to H2O, quenched, did the Timm's stain, then followed with
the rest of the immunostain. No better. The quench is oxidizing the
copper and rendering it undetectable. (Incidentally, the Timm's stain
for other heavy metals is not similarly affected. Presumably the
copper is still affected the same way but the other metals present,
chiefly iron and zinc, survive the quench).
	Question #1:	Is there a way to quench endogenous
			peroxidases without oxidizing? (not likely, I
			realize!) If not...
	Question #2:	Is it possible to reverse the effect of oxidizing
			the copper (i.e. return  the Cu to a form that will
			still reduce Ag in the Timm's stain without
			reactivating endogenous peroxidase).

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
thoughts?

Sorry for the length, but I wanted to explain the problem clearly the
first time. I hope I accomplished that?!
PLEASE post all replies to me AND/OR the Histonet, so all may
benefit. Thanks. Greg
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Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 Unviversity Ave.
Charlottetown, P.E.I.
Canada,  C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851