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Toluidine Blue Staining for Mast Cells

Toluidine blue staining for mast cells post we have briefly explained about Toluidine blue stain method’s principle, requirements, toluidine blue staining procedure, result with Label.

Toluidine Blue Staining

Toluidine blue is a basic thiazine metachromatic dye that has a strong affinity for acidic tissue components, allowing it to stain DNA and RNA-rich tissues. In vivo, toluidine blue staining has been used to detect dysplasia and cancer of the oral cavity. Toluidine blue staining is used in tissue sections to highlight components such as mast cells, granules, mucins, and cartilage.

Principle

The mast cells should stain red-purple (metachromatic staining), while the background should stain blue (orthochromatic staining). The pH, dye concentration, and temperature of the basic dye all contribute to metachromasia, or tissue elements staining a different colour than the dye solution. With metachromatic tissue elements, blue or violet dyes will show a red colour shift, and red dyes will show a yellow colour shift.

Requirements

Chemicals

1. Toluidine blue O

2. Alcohol

3. Sodium chloride

Equipment’s

1. Light Microscope

2. Coplin jars

Reagents

1. Toluidine Blue Stock Solution: toluidine blue staining stock Solution is prepared by dissolving 1 gm of Toluidine blue O in 100 ml of 70% alcohol.

2. Sodium Chloride (1%): Dissolve 0.5 gm of Sodium chloride in 50 ml of double Distilled water. Mix to dissolve (make this solution fresh each time). Adjust pH to 2.0~2.5 using glacial acetic acid or HCl.

3. Toluidine Blue Working Solution (pH 2.0~2.5): Mix 5 ml of Toluidine blue stock solution with 45 ml 1% Sodium chloride, pH 2.3. 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 staining working solution for 2-3 minutes.

3. Wash in distilled water, 3 changes.

4. Dehydrate quickly through 95% and 2 changes of 100% alcohol.

5. Clear in xylene or xylene substitute, 2 changes, 3 minutes each.

6. Coverslip with resinous mounting medium.

Result

Mast cells: Violet/red purple.

Background:  Blue. 

Further Readings

Reference