Mast cell staining [Was untitled]
You'll probably get a lot of different answers to this one!
If you're studying c-kit specifically, then it's a matter of
getting the immunostaining to work.
If you need a stain for mast cells (including young ones)
it may not be necessary to use immunohistochemistry, and
you could save your lab some money by using a dye instead.
For 100 years or so after the first recognition of mast
cells by the great Paul Ehrlich, they were defined as
connective tissue cells with cytoplasmic granules that
stained metachromatically with cationic dyes. From about
the 1950s that translated to granules made of heparin (or
of a similar highly sulphated polysaccharide). It's the
heparin that causes the metachromasia (staining
red with a normally blue dye) and also the affinity for
cationic dyes at pH 1 or lower.
Mast cells stand out prominently in sections stained
with toluidine blue at about pH 4. The background of
blue nuclei is generally helpful. For a selective
stain, alcian blue at pH 1 shows only mast cells,
cartilage matrix and some types of mucus. The mast
cells are easy to see and count with alcian blue, but
the individual granules don't show as sharply as they
do with toluidine blue. You can counterstain after alcian
blue with Mayer's brazalum (that's Mayer's haemalum, but
made with brazilin instead of haematoxylin), which
colours the nuclei of cells red.
A long time ago I used alcian blue and Mayer's brazalum
in an autoradiographic study of the life spans of mast
cells labelled with 3[H]thymidine in baby rats (Journal
of Anatomy 128:225-238, 1979). When the cells first
arrive in the connective tissues they do not contain
stainable heparin (Journal of Anatomy 118:517-529, 1974;
there is also plenty of more recent literature that
establishes haemopoietic tissue as the source of mast
If you need to stain mast cells that are so young that
they don't yet contain heparin granules, the preceding
paragraphs won't help you.
"BORTOLOTTO Susan (SVHM)" wrote:
> If anyone could help, I have just recently started trying to stain mouse mast cells in paraffin embedded tissues with c-kit antibody, and have had no luck, I am getting a very high background and everything seems to stain. Could someone pelase forward me a protocol that is working in their lab. Also, this may not be possible but does anyone know of a way to stain immature mast cells??
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