[Histonet] Re: DAB precipitate

[Patsy Ruegg]

I thought DAB should not be frozen????
Patsy 


Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 215
Aurora, CO 80010
P-720-859-4060
F-720-859-4110
wk email pruegg <@t> ihctech.net
web site www.ihctech.net
 

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Pixley,
Sarah (pixleysk)
Sent: Friday, April 21, 2006 7:18 AM
To: histonet <@t> lists.utsouthwestern.edu
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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