Milligan's trichrome
for Muscle and Collagen
Materials
Solution A
| Material | Amount | |
|---|---|---|
| Potassium dichromate | 2.25 | g |
| Hydrochloric acid, conc | 2.25 | mL |
| Ethanol, 95% | 25 | mL |
| Distilled water | 75 | mL |
Solution B
| Material | Amount | |
|---|---|---|
| Acid fuchsin | 0.1 | g |
| Distilled water | 100 | mL |
Solution C
| Material | Amount | |
|---|---|---|
| Phosphomolybdic acid | 1 | g |
| Distilled water | 100 | mL |
Solution D
| Material | Amount | |
|---|---|---|
| Orange G | 2 | g |
| Phosphomolybdic acid | 1 | g |
| Distilled water | 100 | mL |
Solution E
| Material | Amount | |
|---|---|---|
| Acetic acid, glacial | 1 | mL |
| Distilled water | 100 | mL |
| Directions | ||
Solution F
| Material | Amount | |
|---|---|---|
| Fast green FCF | 0.1 | g |
| Acetic acid, glacial | 0.2 | mL |
| Distilled water | 100 | mL |
Tissue Sample
5µ paraffin sections of neutral buffered formalin fixed were specified. Other fixatives may 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
- Bring sections to water via xylene and ethanol.
- Place into solution A for 5 minute.
- Rinse with distilled water.
- Place into solution B for 5 minutes.
- Rinse quickly with distilled water.
- Place into solution C for 1-5 minutes.
- Rinse with distilled water.
- Place into solution D for 5-10 minutes.
- Rinse with distilled water.
- Place into solution E for 2 minutes.
- Place into solution F for 5-10 minutes.
- Place into solution E for 3 minutes.
- Dehydrate with ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – red
- Erythrocytes – orange
- Muscle – red
- Collagen – green
Notes
- Aniline blue may be substituted for fast green FCF at the same concentration. Collagen would then be blue.
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:
Milligan, (1946)
Technical Bulletin, v. 7, pp.57
