Heidenhain's Azan Trichrome
for Muscle and Collagen

Solutions

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.

Method

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

 

Reference

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