Re: [Histonet] Re: DAB precipitate

From:"Bryan Hewlett"

Hi Patsy,

I have frozen aliquots of DAB for years without any problem!


----- Original Message ----- 
From: "Patsy Ruegg" 
To: "'Pixley, Sarah (pixleysk)'" ; 

Sent: Friday, April 21, 2006 12:14 PM
Subject: RE: [Histonet] Re: DAB precipitate

>I thought DAB should not be frozen????
> Patsy
> Patsy Ruegg, HT(ASCP)QIHC
> IHCtech, LLC
> Fitzsimmons BioScience Park
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> -----Original Message-----
> From:
> [] On Behalf Of Pixley,
> Sarah (pixleysk)
> Sent: Friday, April 21, 2006 7:18 AM
> To:
> Subject: [Histonet] Re: DAB precipitate
> Dear Eva:
> I would like to hear any answers to this also. We always have a DAB
> precipitate. To avoid it, we make the DAB and add the nickel sulfate and
> H2O2 immediately before the reaction and do the reaction quickly. (Using
> nickel in the solution increases the precipitate significantly.) The 
> longer
> you wait, the greater the precipitation. If I am doing a lot of samples, I
> set half the DAB aside and change solutions in the middle (add the nickel
> sulfate and H2O2). Otherwise, I don't know any other tricks.
> One other phenomenon I have seen is after freezing DAB. We make up our DAB
> solution in phosphate buffer (without H2O2 or nickel) and freeze the
> leftover. That works very well as long as you freeze in glass (i.e. new
> glass scintillation vials). If you freeze in some plastics, I have seen a
> precipitate upon thawing. And the glass has to be very clean--it cannot be
> something that was used before for DAB and then bleached and cleaned. Even
> if you clean the glass with a good cleaner like Chromerge or Contrad, 
> there
> can be enough bleach leached into the glass to inactivate the DAB.
> Good luck and I hope to hear some interesting solutions to your problem!
> Sarah Pixley
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