[Histonet] Weak DAB from frozen

[Terri Braud]

I agree with Carl Hobbs: "Perhaps your H2O2 ( substrate) stock has 'gone off'? That will give you a weaker/negative DAB result."
If your frozen DAB aliquots keep the same color, I would be very suspect of the Hydrogen Peroxide.  It can become unstable very quickly.

Terri L. Braud, HT(ASCP)
HNL Laboratories for 
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
Ph: 215-938-3689
Fax: 215-938-2021
Honesty
AccouNtability
AgiLity
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-----Original Message-----
From: histonet-request at lists.utsouthwestern.edu <histonet-request at lists.utsouthwestern.edu> 

Today's Topics:
   1. Re: DAB freezing (Alonso Mart?nez Canabal) (Carl Hobbs)

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Message: 1
From: Carl Hobbs <carl.hobbs at kcl.ac.uk>
Re: [Histonet] DAB freezing (Alonso Mart?nez Canabal)

I do manual IHC-DAB regularly
DAB shouldn't "go off" after freezing unless you have somehow oxidised it during mixing/aliquoting.
It would then show as a dark brown aliquot I state this because I still make up my own DAB from dry powder ( have been doing for > 20yrs) SIgma D5637 I dissolve, aliquot and freeze, as you do.
An aliquot may  be stored at -20C  for 2yrs before I defrost/use it If I run out of 50microL aliquots  I will defrost a 1ml aliquot, re-aliquot into 50 microL ( when I am doing IHC on a few slides, I make up a small working DAB vol.)

Perhaps your H2O2 ( substrate) stock has "gone off"?
That will give you a weaker/negative DAB result

NB: left-over  working DAB solution can be stored at 4C for  at least 7 days and still be perfectly usable ( NOT re-usable) If I have only 5-10 slides I will do the DAB step in the humidity chamber, applying DAB  as I would antibody (only into the hydrophobic pen-ringed section)

Good luck and...please let us know the answer when you solve your problem

Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson SPaRC
Guys Campus, London Bridge
Kings College London
London
SE1 1UL
020 7848 6810


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