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.