Jun 20, 2023

Public workspaceLipid (Oil Red O) Staining

DOI
dx.doi.org/10.17504/protocols.io.5qpvo3n9bv4o/v1
  • 1CeMi
Laura.SabioRodriguez
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.5qpvo3n9bv4o/v1
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 20, 2023
Protocol Integer ID: 83716
Keywords: Lipid droplets, Staining, Adipocytes, Oil Red O
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
Icon for Lipid Oil Red O Staining kit.pdf
Lipid Oil Red O Stai...
226KB
Protocol materials
ReagentIsopropanol
Step 3
ReagentHematoxylin
Step 6
ReagentIsopropanol (IPA) 100%
Before starting
Reagent10 % formalin
Step 2
ReagentDistilled Water
In 4 steps
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 Amount20 mL of ReagentIsopropanol (IPA) 100%Contributed by users . Mix well and leave undisturbed for Duration00:20:00 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 Duration00:10:00 and filter through Whatman No. 1 filter paper. The Working Solution is stable for 2h and must be prepared 15 min before use.
Procedure
Procedure
30m
30m
All components:
Use Amount100 µL / well for a 96 well plate
Use Amount2 mL / well for a 6 well plate
Use Amount6 mL / well for a 100 mm culture dish

Fixation
Remove the medium and gently wash the cells twice with Reagent1X PBS (Phosphate-buffered saline ) . Add Reagent10 % formalin Contributed by users to the cells and incubate for Duration00:30:00 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 ReagentDistilled WaterContributed by users . Add 60% ReagentIsopropanolContributed by users to the cells and incubate for Duration00:05:00 .

5m
Discard 60% isopropanol and cover the cells evenly with Oil Red O Working Solution. Rotate the plate or dish, and incubate for Duration00:10:00 to 20 min .

10m
Discard the Oil Red O Solution and wash the cells 2-5 times with ReagentDistilled WaterContributed by users until no excess stain is seen.

Add ReagentHematoxylinContributed by users to the cells and incubate for Duration00:01:00 . Discard hematoxylin and wash the cells 2-5 times with ReagentDistilled WaterContributed by users .

1m
Cover the cells with ReagentDistilled WaterContributed by users and view under microscope. Lipid droplets appear red and nuclei appear blue.
Note: Keep the cells covered with water to avoid drying.