Jun 21, 2023
Lipid (Oil Red O) Staining
- 1CeMi
- Centre for the Cellular Microenvironment [CeMi]
Protocol Citation: Laura.SabioRodriguez 2023. Lipid (Oil Red O) Staining . protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3n9bv4o/v1
Manuscript citation:
Lipid (Oil Red O) Staining Kit. SIGMA-ALDRICH, Catalog Number: MAK194
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 20, 2023
Last Modified: June 21, 2023
Protocol Integer ID: 83716
Keywords: Lipid droplets, Staining, Adipocytes, Oil Red O, lipid droplet, lipid, present lipid, triglyceride, neutral lipid, present in the adipocyte, adipocyte, adipocytes result, cholesterol ester, organelle, obesity, diabetes
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.
Attachments
Protocol materials
Isopropanol (IPA) 100%
1X PBS (Phosphate-buffered saline )
10 % formalin
Distilled Water
Isopropanol
Hematoxylin
Safety warnings
Hazard Classifications
Acute Tox. 3 Inhalation - Acute Tox. 4 Oral - Carc. 1B - Eye Irrit. 2 - Muta. 2 - Skin Irrit. 2 - Skin Sens. 1 - STOT SE 3
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 20 mL of Isopropanol (IPA) 100% . Mix well and leave undisturbed for 00:20:00 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 00:10:00 and filter through Whatman No. 1 filter paper. The Working Solution is stable for 2h and must be prepared 15 min before use.
Procedure
30m
All components:
Use 100 µL / well for a 96 well plate
Use 2 mL / well for a 6 well plate
Use 6 mL / well for a 100 mm culture dish
Fixation
Remove the medium and gently wash the cells twice with 1X PBS (Phosphate-buffered saline ) . Add 10 % formalin to the cells and incubate for 00:30:00 to 1 h .
Note: Do not add formalin directly onto the cells. Pipette onto the wall and mix gently rotating.
30m
Discard the formalin and wash the cells twice using Distilled Water . Add 60% Isopropanol to the cells and incubate for 00:05:00 .
5m
Discard 60% isopropanol and cover the cells evenly with Oil Red O Working Solution. Rotate the plate or dish, and incubate for 00:10:00 to 20 min .
10m
Discard the Oil Red O Solution and wash the cells 2-5 times with Distilled Water until no excess stain is seen.
Add Hematoxylin to the cells and incubate for 00:01:00 . Discard hematoxylin and wash the cells 2-5 times with Distilled Water .
1m
Cover the cells with Distilled Water and view under microscope. Lipid droplets appear red and nuclei appear blue.
Note: Keep the cells covered with water to avoid drying.