Re: IHC - brown everywhere - Unbrownifying

From:"J. A. Kiernan" <>

Tony H makes a good point. It's very easy to get the hydrogen
peroxide concentration wrong, and the error is always one or 
two orders of magnitude. 100 times too much can brownify
everything, and a hundrendth of what's needed won't work.

This reminds me of something else.

If a DAB solution is brown (like weak tea) it will make
sections even browner without any help from peroxidase etc.
I've not seen this in recent years, but it was commonplace in
the days when DAB was an unreliable reagent. Even in the 1980s
I bought _brown_tablets_ from a highly respected vendor, and
not surprisingly they made a brown solution that stained sections
brown. Pure DAB, which is white and dissolves to make a colourless
solution, has been available for many years. It can be expected to
oxidize on prolonged storage, however, and that's why in many
labs it is weighed out into appropriate aliquots (such as 50 mg)
into capped plastic vials, which are kept in the freezer until
needed. This avoids repeated opening of a stock jar of DAB, with
exposure to fresh air (and perhaps dampness) every time. 

If your DAB is old and makes brown solutions, you can extract the
oxidation products by shaking with activated charcoal and then
centrifuging (or filtering) to obtain a colourless solution. This
is a very simple procedure that costs almost nothing in supplies
and man-hours (Ros Barcelo,A +2. Stain Technol. 64: 97-98; 1989), 
and it lets you use old brown DAB right away - no need to wait a
week for an order of fresh white powder!
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1
On Fri, 20 Apr 2001, Tony Henwood wrote:
> You could try checking your concentration of hydrogen Peroxide in you
> DAB reagent. Too concentrated and you will end up with rapid
> nonspecific DAB deposition.
> Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC)

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