Which mast cells, connective tissue or mucosal, fix/staining

From:Gayle Callis <uvsgc@msu.oscs.montana.edu>

Question:  Which mast cell population are you staining for?  There are two,
with distinct ways of staining for each, including fixation considerations. 

Connective tissue mast cells can be NBF fixed (common in skin, mast cell
tumors) with tissue processing no different than routine tissue work.
These stain very well with Churukians/Schenk's toluidine blue and are
unaffected by decalcification (worked on acid decalcified rat knees) nor
any of the solvents used in routine processing.  I don't think you would
need to use Cedarwood oil.  We always started processing of Carnoys fixed
tissues in 100% alcohol, avoiding lower water gradients (70, 80, 95),
xylene, and paraffin since the tissue is mostly dehydrated already.   

Mucosal mast cells, found in intestinal mucosa particularly after mouse
(our animal model) was infected with a parasitic worm.  These can be
stained for with Astra Blue, very acid pH, and only after Carnoys fixation
(we take chloroform out of Carnoys, so you have an acetic acid/ethanol
fixation.   Mucosal mast cells tend to be smaller, but that is hard to
always determine microscopically. 

However, toluidine blue will stain Carnoys fixed mast cells but astra blue
does not stain NBF fixed mast cells. 

There are publications by Enerbeck Acta path et Microbiol Scandinav 1966,
66:303-312 and Murray M et al, Lab Investigation, 19(2):222-234, 1968 on
mast cell staining, both populations (they vary biochemically), comparing
alcian blue, astra blue, etc with the latter preferred for mucosal mast
cells.   There is also a publication by Patricia Crowle in J of
Histotechnology, 15 or so years ago, on staining of mast cells with these
two stains. 

After this long blab, if you are only looking at  connective tissue mast
cells, go with NBF and routine processing.  Mucosal mast cell studies tend
to be more experimental in nature, but do present a different protocol IF
you want to differentiate them from CTMC plus some clever semiquatitative
design for counting these cells. 



Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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