Re: Mast cell tryptase (Long)

From:"J. A. Kiernan"

Richard Cartun  had asked:
> > Does anyone know if DAKO's mouse anti-human 
> > "Mast cell tryptase" mAb stains canine mast cell 
> > tumors?
Bryan Hewlett replied:
> I do not believe that it does.
> It does not work in mouse or rat tissues in our hands.

I may be a few decades behind the times when it comes to
mast cells, but back in the 1960s and early 1970s they
contained 2 proteolytic enzymes: chymase (chymotrypsin-like)
and tryptase (trypsin-like). These could be detected by
enzyme histochemistry (azo-coupling, with naphthyl amide
substrates, inhibitor controls etc) and were among the few
"tough" enzymes that remained active in paraffin sections.

The chymase was the only proteolytic enzyme in rat and mouse
mast cells. Most other species, including dog and man if I
remember rightly, had both the chymase and the tryptase.
This could explain why anti-(human tryptase) does not give
positive immunostaining of rat or mouse mast cells. If it
doesn't work on dog tissues, the human and dog tryptases
may be too different as antigens despite their biochemical

Bryan H then wrote:
> Have you considered checking for the presence of mast cells 
> using Alcian blue pH 1.0 or the long toluidine blue method?

If your intention is simply to detect mast cells, follow
Bryan Hewlett's advice.  Staining with one of these cationic 
dyes at low pH is 100% reliable, and much less fuss than 
immunohistochemistry. All mast cells have granules composed 
in large part of heparin, which is a sulphated mucosubstance 
and has the expected affinity for cationic dyes. Alcian blue 
at pH 1 is very easy. Toluidine blue is also easy, especially 
if you do it at pH 1, but at a higher pH (eg pH 3-4, which 
will also give blue nuclear  staining), a few practice runs 
are needed to get the metachromasia (red staining by the blue 
dye) of mast cells to resist dehydration and be there in the 
permanent preparation. 

Bryan H also wrote:

> Are they Histamine positive? The mast cells of all 
> mammalian species are histamine positive.

Also true. Amazingly, enough of this small, soluble 
molecule is immobilized by aldehyde fixation to be
detectable in sections. In rodents, the mast cells
contain histamine but most of the effects of mast cell
degranulation are mediated by 5-hydroxytryptamine

***  Another consideration: THE FIXATIVE. 
Rat and mouse mast cell granules are unusual in being
well preserved by aqueous formaldehyde. In many other
species the granules dissolve in aqueous fixatives (and
also in the extracellular fluid in vivo, after being
discharged from the cell). Human mast cell granules are
partly preserved by aqueous fixatives. Dogs have very
soluble heparin, and discharged granules are hard to
find. To preserve mast cells as stainable entities the
optimal fixative is non-aqueous: Carnoy, or an "alcoholic
Bouin," for example. 

There is an abundance of literature about all this, 
mostly more than 40 years old. Fortunately it was all 
thoroughly reviewed by the late Hans Selye in his 
book "The Mast Cells" (1963). They didn't know about
CD antigens then, and the "mucosal mast cell," although
seen and described, was not recognized as a different
and research-worthy subspecies.

Failure to find mast cells after fixation in an aqueous
mixture (except in rats & mice) is just what's expected.
Do you have some similarly processed blocks that ]
certainly contain mast cells?  Anything with loose
connective tissue will have lots. Subcutaneous tissue
and tongue contain many mast cells.

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

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