Re: pale brown DAB reaction; Try UHP

<< Previous Message | Next Message >>
From:"J. A. Kiernan" <>
To:'Histonet' <>
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

  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

<< Previous Message | Next Message >>