Protocol
Crossman's Trichrome
for Muscle and Collagen
Materials
- Solution A
Material Amount Acid fuchsin 0.3 g Orange G 2 g Acetic acid, glacial 1 mL Distilled water 100 mL Thymol a small crystal - Solution B
Material Amount Phosphomolybdic acid 1 g Distilled water 100 mL - Solution C
- Option 1
Material Amount Aniline blue 2 g Acetic acid, glacial 2 mL Distilled water 98 mL - Option 2
Material Amount Light green SF 1 g Acetic acid, glacial 1 mL Distilled water 99 mL
- Option 1
- Solution D
Material Amount Acetic acid, glacial 1 mL Distilled water 99 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.
Protocol
- Bring sections to water via xylene and ethanol.
- Stain nuclei with an acid resistant nuclear stain such as Weigert’s iron hematoxylin.
- Place into solution A for 1 minute.
- Rinse with distilled water.
- Place into solution B until collagen is decolorised.
- Rinse quickly with distilled water.
- Place into solution C for 5 minutes.
- Rinse with distilled water.
- Place into solution D until differentiated.
- Rinse with distilled water.
- Dehydrate with ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – black
- Erythrocytes – yellow
- Cytoplasm – red
- Collagen – blue or green
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.
