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Toluidine Blue Staining Protocol for Mast Cells
Description:
Mast cells are found in the connective tissue and their cytoplasm
contains granules (metachromatic) composed of heparin and histamine.
Toluidine blue should stain mast cells red-purple (metachromatic
staining) and the background blue (orthochromatic staining).
Metachromasia, tissue elements staining a different color from the
dye solution, is due to the pH, dye concentration and temperature of
the basic dye. Blue or violet dyes will show a red color shift, and
red dyes will show a yellow color shift with metachromatic tissue
elements.
Fixation:
10% Formalin.
Sections:
paraffin sections at 5 um.
Solutions and Reagents:
Toluidine Blue Stock Solution:
Toluidine blue O (Sigma) --------------- 1 g
70%
alcohol ------------------------------ 100 ml
Mix
to dissolve.
Sodium Chloride
(1%):
Sodium chloride --------------------------- 0.5 g
Distilled water ----------------------------- 50 ml
Mix
to dissolve (make this solution fresh each time).
Adjust pH to 2.0~2.5 using glacial acetic acid or HCl.
Toluidine Blue
Working Solution (pH 2.0~2.5):
Toluidine blue stock solution -------------- 5 ml
1% Sodium chloride, pH 2.3 --------------- 45
ml
Mix well. The pH should be around 2.3 and less
than 2.5
Make this solution fresh and
discard after use. pH higher than 2.5 will make staining less
contrast.
Procedure:
1.
Deparaffinize and hydrate sections to distilled water.
2.
Stain
sections in toluidine blue working solution for 2-3 minutes.
3.
Wash
in distilled water, 3 changes.
4.
Dehydrate quickly through 95% and 2 changes of 100% alcohol (10 dips
each since stain fades quickly in alcohol).
5.
Clear
in xylene or xylene substitute, 2 changes, 3 minutes each.
6.
Coverslip with resinous mounting medium.
Results:
Mast cells --------------------------- violet/red purple.
Background ------------------------- blue.