[Histonet] Mineral deposits and von Kossa & alizarin S

From:Roger Moretz

A colleague has asked me to post this for him:

We recently found what appears to be mineral within
mouse heart tissue.  This mineral stains with von
Kossa, but was negative with Alizarin Red S, with the
exception of a single mineral deposit in one animal. 
The hearts were fixed in neutral buffered formalin
(NBF).  Is it possible that the calcium leached out in
the NBF and that is the reason for the negative result
with Alizarin Red?  Some of the older literature
suggests using alcohol fixatives, but more recent
literature recommends using NBF.   If the calcium
leached out would you still expect to see non-staining
mineral deposits, or should the entire deposit be
dissolved away and absent from the tissue section?

Also, some people have suggested that to increase the
confidence (of the von Kossa stain) that the blackened
material is calcium a duplicate tissue section can be
decalcified with citrate buffer (pH 4.5), 10 % formic
acid, or 0.5% aqueous hydrochloric acid.  Has anybody
had any luck with one of these procedures to help
characterize mineral?  Is there a reference available
on this modified method?  


Mike Thibodeau, DVM, PhD, DACVP
Senior Scientist, Pathologist
Department of Toxicology and Safety Assessment

Boehringer Ingelheim Pharmaceuticals Inc.
900 Ridgebury Rd, P.O. Box 368
Ridgefield CT, 06877
Phone: (203) 791-6124
Mobile: (203) 546-0260
Email: mthibode@rdg.boehringer-ingelheim.com 

Roger Moretz, Ph.D.
Senior Scientist, Electron Microscopist
Dept of Toxicology & Safety Assessment
Boehringer Ingelheim Pharmaceuticals, Inc.
900 Ridgebury Rd
Ridgefield, CT 06877

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