On Mon, 25 Sep 2000, Bryan Llewellyn wrote:

> There is often an assumption that the tungsten or molybdenum is the culprit,
> although I note you did not say that.  In one of his numerous Picro-Mallory
> stains for fibrin (IV, I think) Lendrum used a solution of trichloroacetic
> acid instead of PTA.  Results were perfectly satisfactory as I recall.
> 
>               ...  In fact I remember reading
> somewhere, sometime that any acid would do ...
> Do you have any comments on this, John?

   Lillie maintained that the separation of cytoplasmic from
   collagen staining could be achieved by low pH alone. He
   didn't seem to like trichromes much, especially those with
   critical differentiations such as AZAN. He favoured variants
   on the van Gieson theme and developed some good methods,
   including picro-aniline blue and its "allochrome" combination
   with PAS. These are all technically simple and are described
   and discussed in "Histopathologic Technic" as well as in 
   Lillie's many papers.

   You certainly cannot replace PTA/PMA with "any acid" in
   Heidenhain's AZAN where it's used alone, not mixed with
   dyes, to displace the last of the red colour from collagen.
   Lillie was well aware of the binding of PMA by collagen
   fibres, which is easily demonstrable by reducing to insoluble
   molybdenum blue (e.g. with SnCl2). This binding is, in fact,
   visible even without making a coloured product. There is
   an obvious change in refractility (if that's a word) in
   collagen in a section that has been immersed in PTA or PMA.

   The most recent mechanistic papers I've seen on connective tissue
   staining are by P. Prento (1993) Histochemistry 99:163-174 and
   the late P. Reid et al. (1993) Histochem. J. 25:821-829.
   Reid came down on the side of a Baker-type explanation,
   corresponding to Horobin's rate-controlled and affinity-controlled
   general mechanisms of staining, with the molecular weight of
   the dyes as an important factor. Prento's approach was different,
   and was confined to Van Gieson's picro-fuchsine. He concluded
   that picric acid attached principally to hydrophobic sites in
   cytoplasm and that hydrogen bonding was important in the binding
   of acid fuchsine to collagen. Both papers are well worth reading
   because they contain much intelligent discussion.

                                        John.
 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1
   Phone: (519) 661-2111
   FAX (Department): (519) 661-3936
   E-mail: kiernan@uwo.ca