Heidenhain's Azan Trichrome
for Muscle and Collagen


Solution A
Azocarmine 2 g
Acetic acid, glacial 1 mL
Distilled water 100 mL
Solution B
Aniline 0.1 mL
Ethanol, 95% 100 mL
Solution C
Hydrochloric acid 0.1 mL
Ethanol, 100% 100 mL
Solution D
Phosphomolybdic acid 5 g
Distilled water 100 mL
Solution E
Orange G 2 g
Aniline blue 0.5 g
Acetic acid, glacial 7.5 mL
Distilled water 100 mL

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. Place into solution A at 50°C for 1 hour.
  3. Rinse quickly with distilled water.
  4. Place into solution B to differentiate nuclei.
    Dip into solution C before examining.
  5. Rinse well with distilled water.
  6. Place into solution D for 2 hours.
  7. Place into solution E for 2-3 hours.
  8. Rinse with distilled water.
  9. Dehydrate with ethanol.
  10. Clear with xylene and mount with a resinous medium.

Expected results


Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.
    Heidenhain, (1905)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
    v.22, pp.339




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