Masson’s Trichrome Staining by Doctor-Dr

Table of Contents

- Masson’s Trichrome Staining definition

- Objectives of Masson’s Trichrome staining

- Principle of the Masson’s Trichrome Staining

- Reagents and Reagent preparation

- Procedure of Masson’s Trichrome Staining

- Results and Interpretation of Masson’s Trichrome Staining

- Applications

- Advantages

- Limitations

Definition of Masson's Trichrome Staining

Masson's Trichrome Staining is a histological staining technique used to specifically stain erythrocytes, muscles, fibrin, and collagen. The name "trichrome" refers to the staining method, which employs three stains. These include aniline blue, Biebrich scarlet-acid fuschin solution, and Weigert's hematoxylin.

Objectives of Masson’s Trichrome staining

• To dye the collage fibres.
• To stain collagen.
• To stain keratin.
• to stain fibrin.
• To dye muscular fibres.

Masson's Trichrome Staining Principle

The Masson's Trichrome staining method makes use of the three stains to identify collagen fibres in tissues including the skin, heart, and muscles. The materials come in frozen sections, paraffin-embedded sections, or formalin-fixed forms. The nuclei are stained with Weigert's Hematoxylin, an iron-based hematoxylin dye. This dye may be stained with acidic solutions without losing colour. Collagen, cytoplasm, muscle, and other acidic tissues are all stained by the Biebrich scarlet-acid fuschin solution. As a decolorizing agent, phosphomolybdic or phosphotungstic acid causes the Biebrich Scarlet-acid fuschin to diffuse out of the collagen fibres. Red stains are left on the muscle cells as a result. Collagen that has had 1% acetic acid added to it is stained with aniline blue to demonstrate the differences across tissue sectiins. With a red backdrop, the nuclei stain black and the collagen fibres blue.

Reagents and preparation for reagents

- Bouin’s Solution (It improves the quality of Masson Trichrome Stain)

• Saturated Picric acid 75 ml
• 40% Formaldehyde 25 ml
• Glacial acetic acid 5 ml

- Weigert’s Iron Hematoxylin Solution

Stock Solution A

• Hematoxylin 1g
• 95% alcohol 100ml

Stock solution B

• 29% Ferric chloride in water 4ml
• Distilled water 95ml

Concentrated Hydrochloric acid 1ml

• Equal parts of stock solution A and B are mixed for use. 
• The mixture is only stable for not more than 3 months.

- Biebrich Scarlet-Acid Fuschin Solution

• 1% aqueous Biebrich Scarlet 90ml
• 1% aqueous Acid fuschin 10 ml
• Glacial acetic acid 1 ml

- Phosphomolybdic-Phosphotungstic Acid Solution

• 5% Phosphomlybdic acid 25ml
• 5% Phosphotungstic acid 25ml

- Aniline Blue Solution

• Aniline blue 2.5g
• Glacial acetic acid 2ml
• Distilled water 100ml

- 1% Acetic Acid Solution

• Glacial acetic acid 1ml
• Distilled water 99ml

Masson's Trichrome Staining Procedure

1. Utilizing 100%, 95%, and 70% alcohol in that order, deparaffinize and rehydrate.
2. Use distilled water to clean.
3. Refix Formalin-fixed tissues in Bouin Solution for a further hour at 56 ° C. The stain's quality is enhanced as a result.
4. To get rid of the yellow hue, rinse under running tap water for five to ten minutes.
5. For 10 min, stain with Weigert's iron hematoxylin solution.
6. Rinse the stain for 10 min under running water.
7. Use distilled water to clean.
8. For 10 to 15 minutes, stain with the Beibrich-Scarlet Acid Fuschin solution.
9. Use distilled water to clean.
10. In the phosphomolybdic-phosphotungstic acid solution, differentiate for 10 to 15 minutes, or until the collagen's red colour fades.
11. Put the stained part in the aniline blue solution and let it sit there for 5–10 minutes to stain.
12. After a quick rinse in distilled water, immerse the stained area for 2–5 minutes in a 1% acetic acid solution.
13. Use distilled water to clean.
14. Dehydrate quickly using 95% ethyl alcohol.
15. In xylene, clear.
16. Utilize a mounting medium to mount.

Results and Interpretation of Masson’s Trichrome Staining

• Collagen fiber stains blue.
• The nuclei stains black.
• The muscles, cytoplasm, and keratin stain red.
• The background stains red.

Applications

• to research muscular diseases such as muscular dystrophy
• to learn about heart diseases including infections and other
• to research hepatic diseases like cirrhosis
• to research kidney diseases including glomerular fibrosis
• to find and examine malignancies on kidney and liver samples.

Advantages

• Compared to the Hematoxylin-Eosin Y Staining technique, it may be utilised to quantify and distinguish the collagen deposition.
• Unlike other stains, it creates distinct representations of the collagen fibre.
• In comparison to most staining methods, it is more accurate.

Limitations

• After preparation, the stock solutions must be used within 24 hours.
• To guard against exposure to the corrosiveness of the utilised chemicals, the staining procedure must be carried out carefully and while wearing protective clothes.