Masson’s Trichrome Staining Protocol for Collagen Fibers

Masson’s trichrome staining protocol for collagen fibers post we have briefly explained about Masson’s trichrome stain principle, requirements, Masson’s trichrome stain procedure, result with label.

Masson's Trichrome Stain

Masson’s trichrome staining protocol for collagen fibers, this technique is employed to detect the presence of collagen fibers within tissues like the skin, heart, and more. On paraffin-embedded, formalin-fixed sections, it is also used to freeze sections. The collagen fibers are stained blue, and the nuclei will be stained black, while the background will be stained red in Masson’s trichrome stain.


Masson’s trichrome stain name suggests that three dyes selectively stain collagen fibers, muscle fibrin, erythrocytes, and muscle. The general rule in trichrome staining is that the less porous tissues are coloured by the smallest dye molecule; whenever a dye of large molecular size is able to penetrate, it will always do so at the expense of the smaller molecule. Others suggest that the tissue is stained first with the acid dye, Biebrich Scarlet, which binds with the acidophilic tissue components. Then when treated with the phospho acids, the less permeable components retain the red, while the red is pulled out of the collagen. At the same time causing a link with the collagen to bind with the aniline blue.


Rinse glassware in DI water

Coplin jars

60°C oven water bath



Weigert’s Iron Hematoxylin Solution

Stock Solution A:

Hematoxylin: 1 g

95% Alcohol: 100 ml

Stock Solution B:

29% Ferric chloride in water: 4 ml

Distilled water: 95 ml

Hydrochloric acid, concentrated: 1ml

Working Solution:

Mix equal parts of stock solution A and B. This working solution is stable for 3 months.

Phosphomolybdic-Phosphotungstic Acid

5% Phosphomolybdic acid: 25 ml

5% Phosphotungstic acid: 25 ml

Bouin’s Solution

Picric acid (saturated): 75 ml

Formaldehyde (37-40%): 25 ml

Glacial acetic acid: 5 ml 

Biebrich Scarlet-Acid Fuchsin Solution

Biebrich scarlet, 1% aqueous: 90 ml

Acid fuchsin, 1% aqueous: 10 ml

Acetic acid, glacial:1 ml

Aniline Blue

Aniline blue: 2.5 g

Acetic acid, glacial: 2 ml

Distilled water: 100 ml

Phosphomolybdic-Phosphotungstic Acid

5% Phosphomolybdic acid: 25 ml

5% Phosphotungstic acid: 25 ml

1% Acetic Acid Solution

Acetic acid, glacial: 1 ml

Distilled water: 99 ml


Wear gloves, goggles, and a lab coat while using Masson’s trichrome stain. Avoid contact and inhalation of dyes and chemicals. Bouin contains: formaldehyde, a known carcinogen, picric acid can become explosive when dry. Toxic by skin absorption. Keep hot uncapped Bouin’s under the hood. Phosphomolybdic, phosphotungstic acid powders, and acetic acid solutions are skin and eye irritants and strong corrosives.


1. Deparaffinize and rehydrate through 100% alcohol, 95% alcohol 70% alcohol.

2. Wash in distilled water.

3. For Formalin-fixed tissue, re-fix in Bouin’s solution for 1 hour at 56C to improve staining quality, although this step is unnecessary.

4. Rinse running tap water for 5-10 minutes to remove the yellow colour.

5. Stain in Weigert’s iron hematoxylin working solution for 10 minutes.

6. Rinse in running warm tap water for 10 minutes.

7. Wash in distilled water.

8. Stain in Biebrich scarlet-acid fuchsin solution for 10-15 minutes. The solution can be saved for future use.

9. Wash in distilled water.

10. Differentiate in the phosphomolybdic-phosphotungstic acid solution for 10-15 minutes or until the collagen is not red.

11. Transfer sections directly (without rinse) to aniline blue solution and stain for 5-10 minutes. Rinse briefly in distilled water and differentiate in 1% acetic acid solution for 2-5 minutes.

12. Wash in distilled water.

13. Dehydrate very quickly through 95% ethyl alcohol, absolute ethyl alcohol (these steps will wipe off Biebrich scarlet-acid fuchsin staining), and clear in xylene.

14. Mount with resinous mounting medium.


Nuclei black

Cytoplasm, muscle, erythrocytes red

Collagen blue

Masson's Trichrome Staining

Masson’s Trichrome Stain

Positive Controls






Light green may be substituted for Aniline blue.

5% phosphotungstic acid for 5 minutes must be substituted when using Light green.

When staining liver biopsies, the collagen is better to light blue than dark blue.

Further Readings