On Mon, 14 Aug 2000 cklein@mail.mdanderson.org wrote:

> Does anyone have a quick and easy to use procedure for the DAB with nickel
> stain?

  It's just as qiick and easy as the regular DAB method.
  For thin sections you can combine the pre-icubation
  and incubation steps. The pre-incubation is desirable
  for thick sections or whole-mounts to allow the largest
  molecules (DAB) to penetrate the tissue thoroughly and
  be where they're needed when the enzyme's substrate
  (hydrogen peroxide) comes along.

  Pre-incubate sections for about 15 minutes at room 
  temperature in the following solution.

    3,3-diaminobenzidine tetrahydrochloride:  25 mg
    Water:  about 2 ml.
    Dissolve the DAB in the water, then make up to
    50 ml with either a 0.1M phosphate buffer or a
    0.05M TRIS buffer, pH 7.2 to 7.4. 
     (If you try to dissolve DAB directly in the
      buffer, it goes cloudy at first, presumably
      due to formation of the free base of DAB, and
      can take 15 m or longer to dissolve completely.)
    2% nickel ammonium sulphate, stock solution in 
      water (keeps for ever):   0.5 ml.

  Add 0.5 ml of 1% hydrogen peroxide and mix well.

  The enzymatic reaction is often complete in 5 min,
  especially if you have pre-incubated with the
  chromogen and buffer as recommended, but you may 
  need to wait for as long as 15 min.

  Wash in water (3 X 1 min).

  Continue as you wish (counterstain, dehydrate etc).
  The reaction product is blue-black and is not extracted
  by water, alcohols or xylene.

  You can substitute 2.5% cobalt chloride for the nickel
  salt, or use a stock solution containing both salts:
  2.5% CoCl2.6H2O and 2% Ni(NH4)2(SO4)2.6H2O. 
 
  The most frequently cited reference for this method is
  J.C.Adams 1981 J Histochem Cytochem 29:775. He wasn't the
  first to do these methods, but he compared nickel with
  cobalt enhancement and recommended using salts of both
  metals. For what it's worth, I don't see much difference
  whichever one (or 2) is used.  With Ni, Co or both the
  result is much more pleasing than weary old DAB-brown,
  and there is greater sensitivity because of the darker
  colour. Red or green counterstains give appropriate
  contrast.

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