Skip to main content

McFarlane’s Trichrome 3rd Variant for Muscle and Collagen

McFarlane's Trichrome 3rd Variant

for Muscle and Collagen



  • Regaud’s iron hematoxylin or equivalent
  • Solution A
    Picric acid1g
    Orange G0.25g
    Ethanol, 95%80mL
    Distilled water20mL
  • Solution B
    Acid fuchsin0.25g
    Ponceau 2R0.25g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Solution C
    Acetic acid, glacial2mL
    Distilled water98mL
  • Solution D
    Picric acid1g
    Phosphotungstic acid10g
    Ethanol, 95%80mL
    Distilled water20mL
  • Solution E
    Aniline blue2.5g
    Acetic acid, glacial2.5mL
    Distilled water97.5mL
  • Solution F
    Picric acid0.5g
    Phosphotungstic acid5g
    Ethanol, 95%20mL
    Distilled water801mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.


  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Régaud’s iron hematoxylin, but do not differentiate with iron alum.
  3. Place into solution A until nuclei are differentiated.
  4. Wash with water until erythrocytes alone remain yellow.
  5. Place into solution B for 5-10 minutes.
  6. Rinse with solution C.
  7. Place into solution D for 5 minutes until differentiated.
  8. Rinse with solution C.
  9. Place into solution E for 10 minutes.
  10. Rinse with solution C.
  11. Place into solution F until differentiated.
  12. Wash with solution C.
  13. Dehydrate with ethanol.
  14. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Erythrocytes  –  yellow
  • Cytoplasm  –  red
  • Muscle  –  red
  • Collagen  –  blue


  • McFarlane also has another multi-step method and a one step method.
  • Solutions A, D and F require dry weights of picric acid. These could be provided from a saturated ethanolic (1 g in 12 mL) solution. Alternate formulas are:

    Solution A

    Picric acid, sat. alc.12mL
    Ethanol, 95%68mL
    Distilled water20mL
    Orange G0.25g

    Solution D

    Picric acid, sat. alc.12mL
    Ethanol, 95%68mL
    Distilled water20mL
    Phosphotungstic acid10g

    Solution F

    Picric acid, sat. alc.6mL
    Ethanol, 95%14mL
    Distilled water80mL
    Phosphotungstic acid5g

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.
    McFarlane, (1944)
    Stain Technology, v. 19, pp. 29