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)
 * 2) *2mL citrate concentrate solution
 * 3) *8mL deionized water
 * 4) Oil Red O Stock Solution
 * 5) *300mg Oil Red O
 * 6) *100mL 99% isopropanol
 * 7) Staining Solution
 * 8) *6 parts of stock Oil Red O
 * 9) *4 parts of distilled water
 * 10) *Allow to stand for 10min
 * 11) *Filter this solution with Whitman paper
 * 12) *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.