Oil Red O 

Oil Red O is a fat-soluble diazol dye, with a maximum absorption at 518 nm. Oil Red O stains neutral lipids and cholesteryl esters but not biological membranes. Oil Red O can be used for detecting and quantifying hepatic steatosis in mouse liver biopsies. Oil Red O staining efficiently helps to visualize the radical changes that occur in tissues as metabolic disease occurs and progresses^[1].

Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)
The complete experimental procedure for Oil Red O (ORO) staining of liposomes ^[2] : 1 Prepare the ORO stock solution 1). Add 500 mg of ORO powder and 100 mL of 100% isopropanol to a 250 mL bottle, and mix thoroughly.
2). After the solution is prepared, it should be stored in an airtight container to prevent exposure to light.
2 Prepare the ORO working solution Dilute the ORO storage solution with water to 60% isopropanol (in a volume ratio of 3:2).
Before use, filter the working solution through a 0.2 μm cellulose acetate sterile syringe filter.
Note: To achieve the best solution quality, it is recommended to prepare the ORO working solution one day in advance and let it stand for overnight. If it needs to be used on the same day, the diluted solution should be mixed on a shaker for at least 2 hours. Before shaking, wrap the centrifuge tube with a paraffin film to prevent leakage.
3 Prepare Samples At a temperature of 20°C, the nematodes were cultured on the nematode growth medium (NGM) inoculated with OP50 Escherichia coli (in the logarithmic late stage) until the target developmental stage was reached.
2). Add 1 mL of PBST solution (containing 0.01% Triton X-100) to the petri dish. Gently shake to separate the nematodes from the culture medium. Incline the petri dish and rinse it with 1 mL of PBST. Transfer the nematode suspension to a 1.5 mL centrifuge tube.
3). Centrifuge at 560 × g for 1 minute. Discard the supernatant and retain the precipitate. Repeat the process by washing with 1 mL of PBST three times. Finally, discard the supernatant and retain only 100 μL.
4). Add 600 μL of 40% isopropanol to the worm precipitate and shake for incubation at room temperature for 3 minutes.
5). Centrifuge at 560 × g for 30 seconds, discard the supernatant and retain only 100 μL. Be careful not to disturb the worm sediment.
Note: If performing high-throughput staining, the volume of PBST should be increased. The washing time of the nematodes in PBST should not exceed 15 minutes. If there are still bacteria in the supernatant, additional washing is required. Incubate with 40% isopropanol using a shaker or oscillator to ensure thorough mixing of the samples.
4 Lipid-Loaded Chromosomes 1). Add 600 μL of ORO working solution to each sample, and invert the centrifuge tube three times to thoroughly mix the nematodes with the staining solution.
2). Incubate at room temperature with rotation at 30 rpm for 2 hours.
3). Centrifuge at 560 × g for 1 minute, then discard the supernatant and retain only 100 μL.
4). Resuspend the sample in 600 μL of PBST and incubate it at 30 rpm for 30 minutes to remove any excess staining solution.
5). Centrifuge at 560 × g for 1 minute, then discard the supernatant and retain only 50 μL.
6). To better distinguish the reproductive cells from the intestinal cell nuclei of nematodes, 1 μL of DAPI can be added to 1 mL of ORO working solution. After staining for 2 hours, the samples can be prepared for imaging. The intestinal cell nuclei are larger, and the reproductive glands can be identified by the developing reproductive cells.
Note: After ORO staining, the nematodes may adhere to the wall of the centrifuge tube and fail to form a clear precipitate. If this occurs, you can perform another centrifugation or let it stand for at least 10 minutes to allow the nematodes to settle.
5 Production
1). Resuspend the worm precipitate in the remaining supernatant and mix thoroughly.
2). Take 5 μL of the nematode suspension and drop it onto a glass slide. Carefully cover the slide to prevent air bubbles.
3). After sealing the edges of the cover slip with nail polish, proceed with the imaging process.
Note: The fixed and stained nematodes may be stiff and difficult to aspirate. It is recommended to cut off the tip of the pipette tip to enlarge the aperture. If imaging cannot be performed immediately, the slide can be stored in a 4°C centrifuge tube rack for up to 24 hours.
6 Imaging
1). Use a microscope camera with color imaging function to capture the ORO-stained nematodes.
2). Use the 5X objective lens to capture the entire field of view of multiple nematodes, then switch to the 10X objective lens to observe the details of individual nematodes.
3). Export the image in TIF format to prevent data loss due to compression.
Note: If using ORO + DAPI staining, switch to fluorescence imaging mode.
7 Result Analysis
1). Import the image into Image J. If the file format is not recognized, it can be read using the Bio-Formats function in the plugin menu.
2). Under the "Stacks" function in the image menu, use "Images-to-Stack" to combine multiple images into a stack for comparison.
3). Adjust the "Brightness/Contrast" settings to enhance the visibility of the lipid droplets.
4). Classify the images based on the accumulation of lipids in the entire body or specific tissues.
5). To assess lipid localization and identify non-specific artifacts, you can select "Stack to RGB" in the "Color" option of the image menu, which will display the signals of the RGB channels respectively.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

408.49

C₂₆H₂₄N₄O

1320-06-5

Solid

Brown to black

OC1=CC=C2C=CC=CC2=C1/N=N/C3=CC(C)=C(/N=N/C4=CC(C)=CC=C4C)C=C3C

Room temperature in continental US; may vary elsewhere.

4°C, protect from light, stored under nitrogen

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Mehlem A, et al. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protoc. 2013 Jun;8(6):1149-54.  [Content Brief]

  [2]. Escorcia W, et al. Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining. J Vis Exp. 2018 Mar 5;(133):57352.  [Content Brief]