Lipid (Oil Red O) Staining
Laura.SabioRodriguez
Abstract
Lipid droplets (LDs) are dynamic, ubiquitously present lipid-storage organelles, predominantly present in the adipocytes. Triglycerides, neutral lipids, and cholesterol esters stored in LDs are the largest sources of energy. The presence of excess LDs in adipocytes results in obesity and obesity-linked pathologies such as dyslipidemia and diabetes type.
Before start
Kit: Lipid (Oil Red O) Staining Kit. SIGMA-ALDRICH, Catalog Number: MAK194
Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents.
Reconstitute the Oil Red O Stock solution with 20mL of 0h 20m 0s to make the Stock Solution, which is stable for 1 year.
The Oil Red O Working Solution is obtained by adding 3 parts of Oil Red O Stock Solution to 2 parts of water. Mix well and leave undisturbed for 0h 10m 0s and filter through Whatman No. 1 filter paper. The Working Solution is stable for 2h and must be prepared 15 min before use.
Attachments
Steps
Procedure
All components:
Use 100µL for a 96 well plate
Use 2mL for a 6 well plate
Use 6mL for a 100 mm culture dish
Fixation
Remove the medium and gently wash the cells twice with 0h 30m 0s.
Note: Do not add formalin directly onto the cells. Pipette onto the wall and mix gently rotating.
Discard the formalin and wash the cells twice using 0h 5m 0s.
Discard 60% isopropanol and cover the cells evenly with Oil Red O Working Solution . Rotate the plate or dish, and incubate for 0h 10m 0s.
Discard the Oil Red O Solution and wash the cells 2-5 times with
Add 0h 1m 0s. Discard hematoxylin and wash the cells 2-5 times with
Cover the cells with
Note: Keep the cells covered with water to avoid drying.
