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.
