Kohashi's Trichrome
for Elastic and Collagen
Materials
Solution A
| Material | Amount | |
|---|---|---|
| Azocarmine | 0.1 | g |
| Acetic acid, glacial | 1 | mL |
| Distilled water | 99 | mL |
Solution B
| Material | Amount | |
|---|---|---|
| Aniline | 0.1 | mL |
| Ethanol, 95% | 100 | mL |
Solution C
| Material | Amount | |
|---|---|---|
| Acetic acid, glacial | 1 | mL |
| Ethanol, 95% | 100 | mL |
Solution D
| Material | Amount | |
|---|---|---|
| Phosphotungstic acid | 5 | g |
| Distilled water | 100 | mL |
Solution E
| Material | Amount | |
|---|---|---|
| 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.
Protocol
- Bring sections to water via xylene and ethanol.
- Place into solution A for 12-15 minutes.
- Rinse quickly with distilled water.
- Differentiate nuclei with solution B.
- Place into solution C for 30-60 seconds.
- Rinse with distilled water.
- Place into solution D for 30-60 minutes.
- Rinse with distilled water.
- Place into solution E for 15-20 minutes.
- Place into 95% ethanol until differentiated.
- Dehydrate with ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – red
- Erythrocytes – orange
- Elastic fibres – purple
- Cytoplasm – red
- Collagen – blue
Notes
- Solution E is Pasini’s trichrome solution.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- 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
