Roque's Stain
for Cell Inclusions
Materials
Stock solution A
Material | Amount | |
---|---|---|
Citrate buffer, pH 5.8 | 100 | mL |
Methyl green, purified | 0.1 | g |
Thionin | 0.0165 | g |
Dissolve the thionin in a small amount of water. Add the buffer and methyl green. Shake and filter. Use fresh.
Citrate buffer, pH 5.8
Material | Amount | |
---|---|---|
Hydrochloric acid, 0.01M | 42 | mL |
Sodium citrate, 0.01M | 58 | mL |
Dehydrant
Material | Amount | |
---|---|---|
Tertiary butanol | 80 | mL |
Ethanol, absolute | 20 | mL |
Tissue Sample
Fix 2mm thick pieces of tissue in 10% formalin containing 1% sodium acetate for 3 hours. Fix smears with methanol.
Protocol
- Bring sections to water via xylene and ethanol.
- Place into the staining solution for 30 minutes at 40°C.
- Rinse briefly with distilled water.
- Place into dehydrant for 30 seconds
- Replace dehydrant, 2 changes, 3 minutes each.
- Rinse with absolute ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – blue-green
- Nucleolar and cytoplasmic basophil substances – red-purple
Notes
- The reference specifies methyl green, but gives the CI number for ethyl green.
- The methyl green should be purified, but as a powder rather than as a solution:
- Add about 10g methyl green to 200 mL chloroform in an Erlenmeyer flask.
- Shake well.
- Filter under vacuum and in a fume chamber.
- Repeat until the chloroform is blue-green instead of violet.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Humason, G. L., (1967).
Animal Tissue Techniques., pp. 278
W. H. Freeman and Company, San Francisco, CA, USA.