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Masson’s Trichrome Original Variants

Masson's Trichrome

Original Variants


Expected Results

  • Nuclei  –  Black (red if an iron hematoxylin was not used)
  • Collagen  –  blue
  • Bone  –  dark blue
  • Epithelia  –  light red
  • Muscle  –  light red
  • Erythrocytes  –  orange
  • Nervous tissue  –  violet


  • Régaud’s iron hematoxylin or equivalent
  • Solution A
    MaterialVar 1Var 2Var 3Var 4
    Acid fuchsin0.5g0.35g1.0g
    Ponceau 2R0.65g1.0g1.0g
    Glacial acetic acid0.5mL1mL1mL1mL
    Distilled water100mL100mL100mL100mL
  • Solution B
    Phosphomolybdic acid1g
    Distilled water100mL
  • Solution C
    Acetic acid, glacial2.5mL
    Distilled water97.5mL
    Aniline blueas required

    Mix the acetic acid and water. Saturate with aniline blue.

  • Solution D
    Acetic acid, glacial1mL
    Distilled water99mL
  • Solution E
    Acetic acid, glacial1mL
    Ethanol, absolute99mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Trichrome stains often benefit from initial Bouin or formal sublimate fixation, or from secondary fixation in Bouin’s fluid for an hour at 56°C.


  1. Bring sections to water via xylene and ethanol.
  2. Optionally, stain nuclei with Régaud’s iron hematoxylin.
  3. Wash in water.
  4. Place into one of the variants of solution A for 5 minutes.
  5. Rinse quickly with water.
  6. Place into solution B for 5 minutes.
  7. Drain, but do not rinse with water.
  8. Place into solution C for 2-5 minutes.
  9. Place into solution D until differentiated.
  10. Dehydrate with solution E.
  11. Clear with salicylic xylene.
  12. Mount with salicylic balsam.


  • Masson also suggested another method.
  • The dye Masson called ponceau 2R is thought to be xylidine ponceau.
  • It was originally considered important to mount sections stained with acid dyes in an acidified mounting medium, in the belief that it preserved brightness and clarity.
  • Salicylic xylene is made by saturating xylene with salicylic acid.
  • Salicylic balsam is made by adding salicylic acid to Canada balsam, then allowing the excess to settle out. The medium should be clear, pale yellow. An alternative is to dip the coverslip into salicylic xylene prior to mounting. The dissolved salicylic acid will then acidify the mounting medium.

Safety Note

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


  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.