Kohashi's Trichrome
for Elastic and Collagen


Solution A
Azocarmine 0.1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Solution B
Aniline 0.1 mL
Ethanol, 95% 100 mL
Solution C
Acetic acid, glacial 1 mL
Ethanol, 95% 100 mL
Solution D
Phosphotungstic acid 5 g
Distilled water 100 mL
Solution E
Eosin B 0.7 g
Acid fuchsin, sat. aqu. 4 mL
Unna's elastic stain 35 mL
Ethanol, 50% 35 mL
Glycerol 40 mL

Tissue sample
Many fixatives are satisfactory. 5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. 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 for 12-15 minutes.
  3. Rinse quickly with distilled water.
  4. Differentiate nuclei with solution B.
  5. Place into solution C for 30-60 seconds.
  6. Rinse with distilled water.
  7. Place into solution D for 30-60 minutes.
  8. Rinse with distilled water.
  9. Place into solution E for 15-20 minutes.
  10. Place into 95% ethanol until differentiated.
  11. Dehydrate with ethanol.
  12. Clear with xylene and mount with a resinous medium.

Expected results


  1. Solution E is Pasini's trichrome solution.


Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.
    Kohashi, (1937)
    Folia anatomica Japonica, v.15, pp.175




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