Jacobs:Protocol Oil Red O Staining

Overview

Protocol for Oil Red O Staining. Useful for identifying adipocytes.

Materials

• Citrate concentrate solution
• Oil Red O
• 99% isopropanol
• Distilled water
• Acetone
• PBS
• 60% isopropanol
• Hematoxylin
• Whitman paper
• Transfer pipets
• Serological pipets/Pipet aid
• Water bath
• Timer
• Waste beaker
• 70% EtOH
• Markers
• Gloves

Solutions

1. Citrate Working Solution (store in 4C for long term use)
   • 2mL citrate concentrate solution
   • 8mL deionized water
2. Oil Red O Stock Solution
   • 300mg Oil Red O
   • 100mL 99% isopropanol
3. Staining Solution
   • 6 parts of stock Oil Red O
   • 4 parts of distilled water
   • Allow to stand for 10min
   • Filter this solution with Whitman paper
   • Working solution is stable for 1-2 hr

Procedure

1. Warm citrate working solution
2. Prepare fixative solution: 2 parts citrate working solution + 3 parts acetone
3. Remove media
4. Wash cells 2x with PBS
5. Fix with 1ml fixative solution for 30sec
6. Wash cells 1x with distilled water for 45sec. Do not allow plates to dry.
7. Stain in 1ml staining solution for 6-15min
8. Clear background using 1ml 60% isopropanol
9. Wash well in distilled water
10. Counter stain with 1ml hematoxylin for 5min
11. Rinse with distilled water

• Lipid: Red
• Nuclei: Blue

Contact

• History: CRJ-EJC, last updated 2/21/07

discuss this protocol.