Roque's Stain
for Cell Inclusions

Solutions

Stock solution A
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
Hydrochloric acid, 0.01M	42	mL
Sodium citrate, 0.01M	58	mL

Dehydrant
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.

Method

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:
  1. Add about 10g methyl green to 200 mL chloroform in an Erlenmeyer flask.
  2. Shake well.
  3. Filter under vacuum and in a fume chamber.
  4. Repeat until the chloroform is blue-green instead of violet.

 

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