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Heidenhain’s Azan Trichrome for Muscle and Collagen

Protocol

Heidenhain's Azan Trichrome

for Muscle and Collagen

10
steps
10
materials

Materials

Solution A

MaterialAmount
Azocarmine2g
Acetic acid, glacial1mL
Distilled water100mL

Solution B

MaterialAmount
Aniline0.1mL
Ethanol, 95%100mL

Solution C

MaterialAmount
Hydrochloric acid0.1mL
Ethanol, 100%100mL

Solution D

MaterialAmount
Phosphomolybdic acid5g
Distilled water100mL

Solution E

MaterialAmount
Orange G2g
Aniline blue0.5g
Acetic acid, glacial7.5mL
Distilled water100mL

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.


Protocol

  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

  • Nuclei  –  Red
  • Muscle  –  Orange
  • Collagen  –  Blue

Safety Note

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


References

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