[Histonet] Re: Mast cells vs eosinophils (LONG)

From:John Kiernan

Expect a lot of replies from Histonet!  Here's
mine for what it's worth.

Toluidine blue at pH 4 will not stain eosinophil
granules - only their nuclei. Mast cell granules
will stain strongly, and if you do the technique
carefully they will be metachromatic (red) even
in the permanently mounted preparation. With less
than perfect technique the mast cell granules will
be purple, or even dark blue. Nuclei of all cells
will also be blue if you stain at pH 4. Cytoplasmic
RNA and mucus will also stain strongly, but they
may not be present at high concentration in
scar tissue. (But what organ? and what sort of scar?)

If you stain at a lower pH (such as 1) cell nuclei
will not stain and mast cell granules may be the
only things visible in the section. You could 
counterstain everything else pink with a dilute
solution of a red anionic dye such as eosin Y.
Eosinophils granules will be stained; they'll be
the only eosinophilic objects if you use an
alkaline solution (pH>8)of the anionic dye.

I've done a lot of mast cell staining. For
quantitative evaluation of intact vs degranulating
cells in rat skin and respiratory tissues my
recommendation is toluidine blue pH 4 because
(a) the light blue background allows you to see
if even a single red granule has been expelled from
the cell, and (b) the presence of the blue nucleus
shows that the section passes roughly through the
centre of the cell. In quantitative work it seems
wise to disregard fragments of mast cells (little
foci of metachromatic granules) that do not have

If you are not worried about mast cells that have
discharged a few granules, and simply want to count 
mast cells that are largely intact, or to see 
generally whether mast cells are present or absent, 
toluidine blue pH 4 is not the best stain. You 
won't want to scan large areas of section at high 

Instead you should stain the mast cells with 
alcian blue at pH 1 or 2.5. Use pH 1 if the
tissue contains mucus or matrix glycuronoglycans
that stain at pH 2.5 and cause confusion. For a
background, stain the cell nuclei red. Mayer's
brazalum (like haemalum, but made with brazilin
instead of haematoxylin) does this very well.

This advice is based on my work with mast cells
of rats and several other species (including man).

Mast cell granules, especially after being 
ejected into the extracellular fluid, vary in
their solubility and fixability. (Have I invented
a new word fixability?)  Dog and guinea-pig mast 
cell granules may be easily lost during fixation.
In rats they hang around as extracellular dots
for many hours. I think humans are rat-like in 
this way, but don't trust my memory. You should
check this up. It's all in Hans Selye's big book,
"The Mast Cells," Washington: Butterworths, 1965.

Your email indicates that you don't want to know
about eosinophils, so I'll refrain from telling
you how to stain them selectively. 
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
rtarmen@drizzle.com wrote:
> Hi everyone,
> I need input on determining between mast and eosinophils. I am staining
> formalin fixed paraffin embedded human scar tissue with acidified
> tol-blue-0 (ph 4). I keep getting increased numbers of violet marked cells
> that I believe are eosinophils, and I only want mast cells with intact
> granulation.
> Any suggestions?
> _cheers,
> Rebecca Armendariz
> Harborview Medical Center
> Univerisity of Washington, Seattle
> rtarmen@drizzle.com
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