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Hollande’s Trichrome for Mitoses, Keratin and Collagen

Hollande's Trichrome

for Mitoses, Keratin and Collagen



  • An alum hematoxylin
  • Solution A
    Basic fuchsin1g
    Ethanol, 70%100mL
  • Solution B
    Hydrochloric acid, conc.0.1mL
    Ethanol, 70%100mL
  • Solution C
    Phosphomolybdic acid1g
    Distilled water100mL
  • Solution D
    Orange Gtosaturation
    Distilled water100mL
  • Solution E
    Light green SF yellowish0.2g
    Distilled water100mL

Tissue Sample

Fixation requirements are unspecified, but likely many fixatives are satisfactory. 5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. Many trichrome stains benefit from Bouin or mercuric chloride fixation of tissue or sections.


  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with an alum hematoxylin nuclear stain and blue.
  3. Place into solution A for 6-12 hours.
  4. Wash with water for 5 minutes.
  5. Place into solution B, a few seconds, until clouds of dye stop being extracted.
  6. Wash well with water.
  7. Place into solution C for 5 minutes.
  8. Rinse with water.
  9. Place into solution D for 5 minutes.
  10. Place into fresh solution E, 30-60 seconds, until differentiated.
  11. Dehydrate with amyl alcohol.
  12. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Mitoses  –  red
  • Cartilage  –  purple
  • Erythrocytes  –  orange
  • Keratin  –  orange
  • Collagen  –  green


  • Amyl alcohol was recommended for dehydration. If this is not available rapid dehydration with ethanol may suffice. Often t-butanol dehydrates satisfactorily without extracting excess dye.
  • The method specified clearing with benzene. For safety reasons this should be avoided. Xylene or toluene should be quite satisfactory.
  • Magenta was specified for solution A. This is an obsolete synonym for basic fuchsin.

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.
    Hollande, (1912)
    Archives de zoologie expérimentale et générale, v.10, pp.62