On Thu, 27 Jan 2000, Robbin Newlin Manager, Histology wrote:

> I am looking for a protacol for enhancing DAB with osmium. Does this
> exist? I thought I had seen it on the Histonet some time ago.

   If this is for electron microscopy your regular post-osmication
   procedure will do the job.

   If its for frozen or vibrating microtome sections or cultured
   cells, for light microscopy, forget it. The OsO4 will darken
   everything unless you rigorously extract all lipids between
   the H2O2-DAB reaction and the osmication. Even if you do this,
   you will still get generalized darkening if you don't wash out
   the OsO4 completely. This means overnight in running water,
   at least, unless the sections are much more than 10um thick.
   It's not worth it.

   If you have mounted paraffin sections, you can put them in an
   aqueous OsO4 solution for about 30 min. The concentration isn't
   critical. 0.5% is OK in my experience. Probably 0.1% would be
   too, if you need a larger volume. From time to time rinse in
   distilled water and check the wet slide with a microscope for
   darkening of the brown polyDAB product and non-darkening of
   areas that you know should be negative. When you're satisfied,
   wash the slides in running tap water for at least 2 hours;
   overnight is better. OsO4 sticks tenaciously and invisibly,
   and any residual traces will eventually darken and spoil the
   preparation. (This advice applies to all LM staining with
   osmium.) Counterstain, wash, dehydrate, clear and cover the
   washed slides in the usual way. The osmication may well
   change the affinities of dyes you use for counterstaining.
   In general, there is an increased affinity for cationic dyes,
   due to oxidative deamination by OsO4, which reduces but
   does not abolish the tissue's attractiveness to anionic dyes.

   Postosmication is a DAB-enhancement method for those who can
   spend time getting the technique right for the purpose at hand.
   There are various enhancement procedures using silver that
   take less time and should give greater amplification (physical
   developer methods). I have not tried any myself; they look quite
   troublesome to do from the published descriptions. Even if you
   buy a kit, you will still need to make visual checks during
   the course of the development.

   If you have not yet done the work, consider icluding nickel
   and/or cobalt ions in the DAB solution. There are lots of
   published recipes, probably all equally good. In the presence
   of these transition metal ions the oxidized DAB polymer is
   a crisp blue-black instead of weary DAB-brown. The different
   colour is an enhancement, but not really an amplification
   although small, weakly positive objects are easier to see.

   The above paragraphs are, I think & hope, by & large true.
   I haven't quoted any references for any of it because they
   are too numerous. Also it's just after midnight and I can't
   be bothered to look them up. So don't feel you have to
   believe any of this. See also a HistoNet email that might
   follow in the next day (or week, or two), about deciding
   which internet utterances you should or should not believe.

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