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Toluidine Blue Staining Protocol for Mast Cells
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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.


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.


      Mast cells --------------------------- violet/red purple.

      Background ------------------------- blue.  

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