Masson's Trichrome Staining Protocol

• Masson's Trichrome Staining is a histological staining method used for selectively stain collagen, collagen fibers, fibrin, muscles, and erythrocytes. It uses three stains for staining hence the term Trichrome. These are Weigert's Hematoxylin, Biebrich scarlet-acid fuchsin solution and Aniline blue.
• By use of the three stains, Masson's Trichrome staining technique is used for the detection of collagen fibers in tissues such as the skin, heart, muscles. The samples are formalin-fixed, paraffin-embedded sections or frozen sections.

• Preparation of the Bouin's solution. Mix 75 ml Saturated Picric acid, 25 ml 40% formaldehyde and 5 ml glacial acetic acid.
• Preparation of Weigert's iron hematoxylin solution. Stock solution A is composed of 1 g hematoxylin and 100 ml 95% alcohol. Stock solution B is composed of 4 ml 29% ferric chloride in water, 95 ml distilled water and 1 ml concentrated hydrochloric acid. Equal parts of stock solution A and B are mixed for use. The mixture is only stable for not more than 3 months.
• Preparation of Biebrich Scarlet-Acid Fuschin solution. Mix 90 ml 1% aqueous Biebrich Scarlet, 10 ml 1% aqueous acid fuschin and 1 ml glacial acetic acid.
• Preparation of phosphomolybdic-phosphotungstic acid solution. Mix 25 ml 5% phosphomlybdic acid and 25 ml 5% phosphotungstic acid.
• Preparation of aniline blue solution. Mix 2.5 g aniline blue, 2 ml glacial acetic acid and 100 ml distilled water.
• Preparation of 1% acetic acid solution. Mix 1 ml glacial acetic acid and 99 ml distilled water.
• Deparaffinize and rehydrate using 100% alcohol, 95% alcohol, and 70% alcohol sequentially. Wash in distilled water.
• For tissues fixed with Formalin, refix in Bouin Solution for 1 hour at 56°C. This improves the quality of the stain. Rinse with running tap water for 5-10 minutes to remove yellow color.
• Stain with Weigert's iron hematoxylin solution for 10 minutes. Rinse the stain with running tap water for 10 minutes. Wash in distilled water.
• Stain with the Beibrich-Scarlet Acid Fuschin solution for 10-15 minutes. Wash in distilled water.
• Differentiate in the phosphomolybdic-phosphotungstic acid solution for 10-15 minutes or until the collagen loses its red color.
• Transfer the stained section to aniline blue solution and stain for 5-10 minutes.
• Rinse the stained section briefly in distilled water and differentiate with 1% acetic acid solution for 2-5 minutes. Wash in distilled water.
• Quickly dehydrate through 95% ethyl alcohol. Clear in xylene.
• Mount with a mounting medium.

• The stock solutions must be used within 24 hours of preparation.
• The stain must be performed with caution and while wearing protective clothing to avoid exposure to the corrosiveness of the reagents used.
• It can be used to measure and differentiate the collagen deposition as compared to the Hematoxylin-Eosin Y Staining method.
• It is more accurate than most staining techniques.

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