Masson's Trichrome
for Muscle and Collagen

Solutions

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

Method

  1. Bring sections to water via xylene and ethanol
  2. Stain nuclei with Weigert's iron hematoxylin or equivalent.
  3. Wash well in tap water, rinse with distilled water.
  4. Place into solution A for 10 minutes.
  5. Rinse with distilled water.
  6. Place into solution B for 5 minutes.
  7. Rinse with distilled water.
  8. Place into solution C for 10 minutes.
  9. Rinse well with distilled water.
  10. Dehydrate with Ethanol.
  11. Clear with xylene and mount with a resinous medium.

Expected results

Notes

  1. Since Masson gave details of his original method, there have been large numbers of 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.

Reference
The method described is in common use in many laboratories, and the details given are based on details in:–
Theory and practice of histological techniques,
Bancroft, J. D. and Stevens, A.
Churchill Livingstone, London, England

 


 

 

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