Kohashi's Trichrome
for Elastic and Collagen

Solutions

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.

Method

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

• Nuclei  –  red
• Erythrocytes  –  orange
• Elastic fibres  –  purple
• Cytoplasm  –  red
• Collagen  –  blue

Notes

1. Solution E is Pasini's trichrome solution.

 

Reference

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