Re: pale brown DAB reaction; Try UHP
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | 'Histonet' <histonet@pathology.swmed.edu> |
Reply-To: | |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
Here are a few ideas that relate to the hydrogen peroxide
part of Geoff's message.
On Fri, 14 Apr 2000, Geoff McAuliffe wrote:
> Sounds like your DAB or peroxide are weak. Try fresh, as in buy a new
> bottle, reagents. I have known DAB to work well one week and fail the next.
> Also peroxide turns to water over time. In my lab, both of these reagents get
> replaced every year. Cheap insurance.
Instead of 30% hydrogen peroxide (which can and does turn to water,
and always unexpectedly), you can use a white powder called
UREA HYDROGEN PEROXIDE as a stock of H2O2. It is an addition
compound, to be treated as approximately 35% H2O2, and it is sold
by various chemical vendors (Sigma and Polysciences if I remember
rightly, and surely others too). The label on the rather large
jar (it's not expensive) says keep at 4C. I've done this with my
first purchase of urea hydrogen peroxide (about 1995). It's
still working fine, and there's enough left to last another 5
years if it remains stable in the solid form.
I dig out and weigh 1.43 grams of UHP, using an ordinary nickel
spatula, and dissolve it in 50 ml of pure water, in a very clean
glass-stoppered bottle that's kept for this purpose.
Hydrogen peroxide decomposition is catalyzed by all forms of dirt,
including many metal ions. A nickel spatula seems harmless, but I
know from experience that contact with a hypodermic needle (steel)
can kill a whole 500 ml bottle of 30% H2O2 in less than a week.
A hard plastic spatula may be OK, as may a bottle with a plastic
stopper, especially if the plastic is something chemically inert
like teflon. Slow decomposition occurs even when you haven't (or
don't think you have) contaminated the stock bottle. Phosphate
ions are said to accelerate the self-oxidation/reduction of
H2O2.
1.43 g of UHP in 50 ml water makes an approximately 1% solution, and
this can be diluted appropriately for use in any histochemical
method for peroxidase activity. I don't know how long the 1% H2O2
can be kept for. The longest I have kept it is 3 weeks; but why
not err on the side of caution and make a new stock 1% H2O2 every
week? Your jar of urea hydrogen peroxide will generate enough
throw-away 1% bottles to last several months even in a busy lab.
I learned about UHP from some junk-mail advertising several years
ago, and this is one instance for which I feel grateful, and am
glad I didn't throw the unopened envelope into the bin.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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