Masson’s Trichrome Staining – Principle, Procedure, Result, Uses

Trichrome stains, such as Masson’s Trichrome, derive its name from the three dyes utilised in the staining process. These three dyes are used to selectively stain muscle, collagen fibres, fibrin, and erythrocytes using acid-base chemistry. Initial placement of tissue slices in Bouin’s solution. As a mordant, Bouin’s solution links the dye to the specific tissue components. Weigert’s hematoxylin, an iron hematoxylin that is resistant to decolorization by subsequent acidic staining solutions, is used to stain nuclei. The solution of Biebrich scarlet-acid fuchsin stains all acidophilic tissue components, including cytoplasm, muscle, and collagen. As a decolorizer, the subsequent application of phosphomolybdic/phosphotungstic acid causes the Biebrich scarlet-acid fuchsin to diffuse out of the collagen fibres. This causes the muscle cells to become reddened. The phosphomolybdic/phosphotungstic acid also acts as a link between the decolored collagen and aniline blue, which is administered following the differentiation step. After tissue sections have been stained with aniline blue, 1% acetic acid is added to distinguish them.

What is Masson’s Trichrome Staining?

Histology utilises Masson’s trichrome, a three-color staining procedure. The formulations derived from Claude L. Pierre Masson’s (1880–1959) original formulation have various particular applications, but they are all suitable for differentiating cells from surrounding connective tissue. Connective tissues come within the category of ‘trichrome stains’ despite the availability of numerous procedures for distinction. The phrase “trichrome stain” refers to the use of three dyes, one of which is a nuclear stain that allows for the selective demonstration of muscle, collagen fibres, fibrin, and erythrocytes. In tissue slices, this approach is used to distinguish between smooth muscle and collagen fibres. The vast majority of recipes yield red keratin and muscle fibres, blue or green collagen and bone, light red or pink cytoplasm, and dark brown to black cell nuclei.


The trichrome is applied by immersing the fixed specimen in Weigert’s iron hematoxylin, followed by the following three solutions, designated A, B, and C:

  • Weigert’s hematoxylin consists of three solutions: ferric chloride in diluted hydrochloric acid, hematoxylin in 95% ethanol, and potassium ferricyanide solution alkalinized with sodium borate. It is employed to stain nuclei.
  • Solution A contains acid fuchsin, Xylidine Ponceau, glacial acetic acid, and distilled water; it is also known as plasma stain. Other red acid dyes, such as Biebrich scarlet, can be used in Lillie’s trichrome.
  • Solution B is composed of phosphomolybdic acid dissolved in distilled water.
  • Solution C, often known as fibre stain, comprises yellowish Light Green SF or Fast Green FCF. It is utilised to colour collagen. If blue is desired instead of green, methyl blue or water blue might be used as a substitute.

Principle of the Masson’s Trichrome Staining

Utilizing the three stains, Masson’s Trichrome staining is used to detect collagen fibres in tissues such as skin, heart, and muscles. The specimens consist of formalin-fixed, paraffin-embedded, or frozen sections. To stain the nuclei, Weigert’s Hematoxylin, an iron hematoxylin dye, is utilised. This colourant is resistant to bleaching by acidic staining solutions. The scarlet-acid fuschin solution of Biebrich stains all acidic tissues, including cytoplasm, muscle, and collagen. As a decolorizing agent, phosphomolybdic or phosphotungstic acid is utilised, causing the Biebrich Scarlet-acid fuschin to diffuse out of the collagen fibres. This causes the muscle cells to become stained red. Aniline blue is used to stain collagen along which 1% acetic acid is added to differentiate between tissue sections. With a red background, the collagen fibres stain blue and the nuclei stain black.


Reagents and Reagent preparation

Biebrich Scarlet-Acid Fuchsin Solution

Bibrich scarlet2.25 gm
Acid fuchsin0.25gm
Acetic acid, glacial2.5 gm
Distilled water250 ml

1% Phosphomolybidic Acid Solution

Phosphomolybdic acid 2.5 gm
Distilled water250 ml

1.8 % Aniline Blue Solution

Aniline blue, C.I. 42755 4.5 gm
Acetic acid, glacial4.5 ml
Distilled water250 ml

1% Acetic Acid Solution

Acetic acid, glacial 2.5 ml
Distilled water250 ml

Acid-Alcohol Differentiation Solution

0.5% hydrochloric acid in 70% alcohol

Weigert’s Iron Hematoxylin Solution

Stock Solution A:

Hematoxylin1 g
95% Alcohol100 ml

Stock Solution B:

 29% Ferric chloride in water4 ml
Distilled water95 ml
Hydrochloric acid, concentrated1ml

Weigert’s Iron Hematoxylin Working Solution Preparation: Combine stock solutions A and B in equal proportions. This solution is stable for three months (no good after 4 months)


Procedure of Masson’s Trichrome Staining

  1. Deparaffinize and hydrate the histology section until you achieve distilled water.
  2. Place six drops of Reagent A onto the mixture.
  3. Add six drops of the Reagent B. Allow 10 minutes for a response.
  4. Drain the solution and add 10 drops of Reagent C without washing. Allow 4 minutes for a response.
  5. Rinse for three to four seconds with pure water.
  6. Dispense 10 drops of Reagent D. Allow four minutes for a reaction time.
  7. Rinse with water that has been distilled.
  8. Distribute ten drops of Reagent E. Allow 10 minutes for a response.
  9. Drain the solution and add 10 drops of Reagent F without washing. Allow 5 minutes for a reaction time.
  10. Rinse with distillated water.
  11. Dehydrate using a succession of increasing alcohols.
  12. Immerse for one minute in pure ethanol.
  13. Xylene should be used for rinsing.
  14. Mount using a mounting medium.
  15. Observe under a microscope.

Results and Interpretation of Masson’s Trichrome Staining

Nuclei and gamets Black
Cytoplasm, keratin, muscle fibers, acidophilic granulationsRed
Collagen, mucus, basophilic pituitary granulationsBlue
Hypophysis delta cell granulesBlue-violet
Rat airway stained with Masson's trichrome The connective tissue is dyed blue, while the nuclei and cytoplasm are stained dark red/purple and red/pink, respectively.
Rat airway stained with Masson’s trichrome The connective tissue is dyed blue, while the nuclei and cytoplasm are stained dark red/purple and red/pink, respectively.
Mouse skin stained with Masson's trichrome stain.
Mouse skin stained with Masson’s trichrome stain.

Applications of Masson’s Trichrome Staining

Masson’s Trichrome staining is a popular staining method used in histology and pathology for the visualization of connective tissue, muscle fibers, and cells in biological tissues. Here are some of the applications of Masson’s Trichrome staining:

  1. Assessment of tissue fibrosis: Masson’s Trichrome staining can help to assess the degree of fibrosis in tissues, which can be helpful in determining the stage of chronic diseases such as liver cirrhosis, kidney fibrosis, and pulmonary fibrosis.
  2. Evaluation of muscle tissue: Masson’s Trichrome staining is used to evaluate muscle fibers and assess the degree of muscle damage in diseases such as muscular dystrophy, myocarditis, and rhabdomyolysis.
  3. Diagnosis of tumor tissue: Masson’s Trichrome staining can be used to diagnose the type of tumor tissue, such as sarcoma or carcinoma.
  4. Study of cardiovascular disease: Masson’s Trichrome staining can be used to study the development of cardiovascular disease, including the evaluation of the formation of fibrous tissue in the heart.
  5. Study of degenerative diseases: Masson’s Trichrome staining can be used to study degenerative diseases such as Alzheimer’s disease and Parkinson’s disease by assessing changes in the structure of the brain tissue.

Overall, Masson’s Trichrome staining is a valuable tool for the analysis of tissue samples in the fields of histology, pathology, and biomedical research.


Advantages of Masson’s Trichrome Staining

  • Compared to the Hematoxylin-Eosin Y Staining technique, it can be used to quantify and discriminate collagen deposition.
  • Compared to other stains, it provides images of the collagen fibre that are more distinct.
  • It is more precise than the majority of staining methods.

Disadvantages of Masson’s Trichrome Staining

  • The stock solutions must be utilised within twenty-four hours of their preparation.
  • To avoid exposure to the corrosiveness of the utilised reagents, the stain must be conducted with caution and while wearing protective equipment.

Important Notes

  • The microscope should meet the specifications of a clinical diagnostic laboratory. If an automatic staining equipment is utilised, the manufacturer’s operating instructions and software must be followed.
  • All samples should be handled in accordance with current technological standards. All samples must be labelled explicitly.
  • Diagnosis should only be determined by authorised and trained individuals. Each application must include appropriate controls to eliminate the possibility of erroneous outcomes.
  • The staining solution should be stored between +15 and +25 degrees Celsius.
  • The product stored at the specified temperature and in a firmly sealed container remains useful until the designated expiration date.
  • To prevent mistakes, staining must be performed by trained professionals. Only for professional use. National directives on workplace safety and quality assurance must be followed.


  • Normal precautions should be taken when handling laboratory reagents.
  • Only competent and trained professional users should utilise this product
  • The product, which contains alcohol and is classed as extremely flammable, must be stored away from sources of ignition.
  • It can cause significant irritation to the eyes and skin. Refer to the Material Safety Data Sheet for latest information on risks, hazards, and safety.
  • Dispose of waste in accordance with all applicable local, state, provincial, and national laws.
  • Reagents should not be used after their expiration date
  • Wear protective clothes and gloves when working with chemicals.
  • Prevent microbiological contamination of reagents, as it may result in inaccurate results.


What is Masson’s Trichrome staining?

Masson’s Trichrome staining is a histological staining method used to visualize the different components of biological tissues, such as connective tissue, muscle fibers, and cells.

Why is Masson’s Trichrome staining used?

Masson’s Trichrome staining is used to assess the degree of fibrosis in tissues, evaluate muscle fibers, diagnose tumor tissue, study cardiovascular disease, and study degenerative diseases.


What type of tissue can Masson’s Trichrome staining be used on?

Masson’s Trichrome staining can be used on a wide range of tissues, including liver, kidney, lung, muscle, heart, brain, and tumor tissue.

How does Masson’s Trichrome staining work?

Masson’s Trichrome staining uses a combination of dyes to differentiate the different components of a tissue sample. The dyes are absorbed into the tissue and then visualized under a microscope.

What dyes are used in Masson’s Trichrome staining?

The dyes used in Masson’s Trichrome staining include aniline blue, acid fuchsin, and light green.

Is Masson’s Trichrome staining reliable?

Masson’s Trichrome staining is a well-established and reliable staining method, widely used in histology and pathology for the analysis of tissue samples.

How long does Masson’s Trichrome staining take?

Masson’s Trichrome staining usually takes 2-3 hours, depending on the specific protocol being used.

Can Masson’s Trichrome staining be performed on fixed tissue samples?

Yes, Masson’s Trichrome staining is typically performed on fixed tissue samples, such as those preserved in formalin.

What are the limitations of Masson’s Trichrome staining?

The limitations of Masson’s Trichrome staining include the potential for tissue over-staining or under-staining, as well as the need for specialized equipment, such as a microscope, to visualize the stained tissue.

How can the results of Masson’s Trichrome staining be interpreted?

The results of Masson’s Trichrome staining can be interpreted by a trained histologist or pathologist, who will analyze the stained tissue sample under a microscope and determine the degree of fibrosis or muscle damage, or identify the type of tumor tissue present.



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