Skip to main content

Roque’s Stain for Cell Inclusions

Roque's Stain

for Cell Inclusions

7
steps
6
materials

Materials

Stock solution A

MaterialAmount
Citrate buffer, pH 5.8100mL
Methyl green, purified0.1g
Thionin0.0165g

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

MaterialAmount
Hydrochloric acid, 0.01M42mL
Sodium citrate, 0.01M58mL

Dehydrant

MaterialAmount
Tertiary butanol80mL
Ethanol, absolute20mL

Tissue Sample

Fix 2mm thick pieces of tissue in 10% formalin containing 1% sodium acetate for 3 hours. Fix smears with methanol.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 30 minutes at 40°C.
  3. Rinse briefly with distilled water.
  4. Place into dehydrant for 30 seconds
  5. Replace dehydrant, 2 changes, 3 minutes each.
  6. Rinse with absolute ethanol.
  7. 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

  1. Humason, G. L., (1967).
    Animal Tissue Techniques., pp. 278
    W. H. Freeman and Company, San Francisco, CA, USA.