Masson's Trichrome

for Muscle and Collagen

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent.
Solution A
Material Amount
Acid fuchsin 0.5 g
Xylidine ponceau 0.5 g
Distilled water 99 mL
Glacial acetic acid 1 mL
Solution B
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution C
Material Amount
Light green SF yellowish 2 g
Glacial acetic acid 2 mL
Distilled water 100 mL

Material	Amount
Acid fuchsin	0.5	g
Xylidine ponceau	0.5	g
Distilled water	99	mL
Glacial acetic acid	1	mL

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Material	Amount
Light green SF yellowish	2	g
Glacial acetic acid	2	mL
Distilled water	100	mL

Tissue Sample

5 µ paraffin sections of Bouin, formal sublimate or B5 fixed tissues are preferred. Zenker and Helly fixation is usually satisfactory. Formalin variants are generally adequate, but staining can be improved by secondary fixation of cut sections with Bouin’s fluid.

Protocol

Bring sections to water via xylene and ethanol.
1. If formalin fixed, refix in Bouins fluid for one hour at 56°C
2. Wash well in tap water to remove the yellow colour.
Stain nuclei with Weigert’s iron hematoxylin or equivalent.
Wash well in tap water, rinse with distilled water.
Place into solution A for 10 minutes.
Rinse with distilled water.
Place into solution B for 5 minutes.
Rinse with distilled water.
Place into solution C for 10 minutes.
Rinse well with distilled water.
Dehydrate with Ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Cytoplasm – red
Erythrocytes – red
Muscle – red
Collagen – green
Nuclei – dark brown

Notes

Since Masson gave details of his original method, there have been numerous variations published. One common variant is Lillie’s, a method often erroneously called “Masson’s” trichrome. These variants differ in the specifics of the dyes used, the concentration of the dyes and polyacid and the times for which they are applied.
The times given here should be considered a guide, although they will generally be found satisfactory if the procedure is followed completely, including refixation of sections in Bouin’s fluid.
Refixing the sections in Bouin’s fluid intensifies the colours and increases the contrast between the tissue components. It is sometimes incorrectly referred to as mordanting, but it is simply a form of secondary fixation of sections and has no real mordanting effect. It should be noted that primary fixation in Bouin’s fluid is recommended for this method.
The dyes in solution A stain the muscle, fibrin, cytoplasm and erythrocytes and the solution is sometimes referred to as the plasma stain. Acid fuchsin and xylidine ponceau are the dyes originally recommended. Many other red acid dyes have been suggested. Biebrich scarlet, for instance, being used by Lillie.
Solution C is sometimes called the fibre stain. It is used to colour collagen. Some other materials will also be coloured, but the method is not used to demonstrate them. Light green SF yellowish is commonly used, but fast green FCF gives a more emerald green. This is preferred by some microscopists. Although this latter dye is considered less likely to fade, it is not as commonly used.
Aniline blue, or either of its constituents, methyl blue, or water blue may be substituted alone or in combination if blue stained collagen is preferred to green.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

The method described is in common use in many laboratories. A typical description would be as found in:

Theory and practice of histological techniques,
Bancroft, J. D. and Stevens, A.
Churchill Livingstone, London, England

Masson’s Trichrome for Muscle and Collagen