On Tue, 29 Jun 1999, Rebecca Hartley wrote:

> I used Sigma's metal-enhanced DAB tablets on 40 um free-floating sections
> (after primary antibody and ABC elite kit), followed by a counterstain with
> Mayer's Hematoxylin, rinses in ddH20 and aqueous mounting in Biomeda's
> Gel/Mount for aqueous and dry mounting.
>
> Here's the problem: Immediately after the DAB step, the staining looked very
> strong.  However, after the hematoxylin step the intensity of staining had
> decreased, and one day after mounting the sections were light brown
> instead ...

   Something has to be very wrong here. According to the Sigma catalog
   (p. 1471) the metal is Co. It looks as if your alum-haematoxylin is
   removing Co from the reaction product, leaving behind the brown
   metal-free oxidation product of DAB. You would think many other
   people would have had this trouble too. The stain is quite
   strongly acidic, with a high concentration of Al ions. Perhaps
   such a mixture should be avoided.

   Alum-haematoxylin may be an unconventional choice of counterstain
   because blue nuclei do not provide optimum contrast for a
   blue-black principal reaction product. Your use of an aqueous
   mounting medium is also unusual when both the stains resist
   dehydration and clearing, but from what you write it's the
   counterstain that's causing the trouble first, with
   decolorization continuing more slowly in the mountant.

   If you must use the Co-DAB tablets I would recommend counterstaining
   with a red basic dye from an only slightly acidic solution.
   0.5% neutral red, adjusted to pH 4.0, is generally very
   reliable. It will stain some other things in addition to nuclei,
   but that may not matter or may even be advantageous. If the
   red colour is too strong you can weaken it by immersion in
   70% alcohol. ***Make a quick check under a microscope before
   applying a coverslip.***  You cannot use an aqueous mountant
   because this will extract a basic dye like neutral red. When
   the colour is right, complete the dehydration in 3 changes of
   100% alcohol, clear in 2 lots of xylene and apply a
   coverslip using an ordinary resinous mounting medium.

   It would be wise to make preliminary trials with sections
   of something less precious than your research material.

   Hope this helps. I wonder if anyone else has had trouble
   with the Cobalt-DAB product and alum-haematoxylin
   counterstaining? If so, it needs to be investigated.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1