Masson's Trichrome Stain



1. Objective

The objective of this test was to use Masson's Trichrome staining to differentiate between muscle, collagen fibers, fibrin, and erythrocytes in tissue sections for histopathological analysis.

2. Principle

Masson's Trichrome stain utilized a three-dye system that differentially stained tissue components based on their chemical properties:

  • Nuclei were stained with a basic dye (Weigert’s iron hematoxylin)

  • Cytoplasm, muscle, and keratin were stained red with Biebrich scarlet-acid fuchsin

  • Collagen fibers and mucin were stained blue or green depending on the aniline blue or light green counterstain

This enabled clear distinction between fibrous connective tissue and muscle, useful in assessing fibrosis or scarring.

3. Materials

  • Formalin-fixed, paraffin-embedded tissue sections

  • Weigert’s iron hematoxylin

  • Biebrich scarlet-acid fuchsin

  • Phosphomolybdic/phosphotungstic acid

  • Aniline blue (or light green)

  • Acetic acid (1%)

  • Deparaffinization reagents (xylene, alcohols)

  • Mounting medium and coverslips

  • Slide racks, glassware, microscope

4. Procedure

  • Sections were deparaffinized and rehydrated.

  • Stained in Weigert’s iron hematoxylin for nuclei (10 minutes).

  • Washed and stained in Biebrich scarlet-acid fuchsin for cytoplasm and muscle (10–15 minutes).

  • Treated with phosphomolybdic/phosphotungstic acid for differentiation.

  • Stained in aniline blue for collagen (5–10 minutes).

  • Washed in 1% acetic acid, dehydrated, cleared, and mounted.

5. Result (Example)

Tissue Component Color
Nuclei Black/blue-black
Muscle fibers, cytoplasm Red
Collagen fibers Blue (or green)
RBCs Bright red

  • Example: Liver biopsy showed blue collagen fibers in fibrotic bands consistent with early-stage cirrhosis.

6. Uses

  • Assessed fibrosis, especially in liver, kidney, and muscle

  • Distinguished between smooth muscle and connective tissue

  • Evaluated tumor stroma and vascular walls

  • Commonly used in liver biopsies, myocardium, and renal pathology

7. Conclusion

Masson's Trichrome staining was an essential histochemical method for evaluating fibrotic changes and muscle structure, providing detailed contrast between tissue elements.

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