[Histonet] DAB (to freeze or not to Freeze)

[Brian Chelack]

Our lab freezes down aliquots of DAB concentrate (typically a 50 mg/ml
concentrate containing 0.1% Brij-35 and Tween -20 is frozen in 100 uL
volumes in tiny eppendorf style plastic vials). We do this to minimize the
handling and thus potential for contact with the DAB. A single 1 gram vial
of dab is added to 20 mls of buffer in a closed 50 ml tube and mixed until
dissolved. We run the concentrate through 0.45 um syringe filter prior to
freezing to remove any insolubles. We thaw the vial when required, add it to
10 mls of buffer add the H2O2 and use. Occasionally we see a bit of ppt.
when the concentrate is thawed, but this dissolves again easily when the
concentrate is added to the buffer. The frozen concentrate is stable for at
least 6 months, and it is convenient. The small volume of concentrate thaws
in just a minute or so. 

 

My experience with DAB has been that the DAB sold by different manufacturers
has slight impurity differences. This provides for different shades of
brown. Most of them seem to work. Personally I don't think it matters which
one you choose, as long as you like the colour.  I've done the Ni and even
Co, and they seem a bit more sensitive, although this might just be due to
the different coloured reaction product, but really haven't been worth the
bother. I've always found they also increase the overall background in a
stain and the important part of the chromagen development is the ratio of
the "signal" you want to the "noise" you don't want. If you need more
sensitivity, generally the best way to achieve it is through your
amplification method. When it comes to chromagens; keep it simple and keep
it consistent, otherwise you run the risk of driving the slide evaluator
nuts with the constant day to day variations

 

We have been very happy with the DAB we purchase from EMS. It has been very
consistent through numerous purchases over the past almost 20 years (man,
I've been doing this way too long.)

 

Brian Chelack

Prairie Diagnostic Services

Saskatoon, SK