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].

Complete protocol for staining liposomes with Oil Red O (ORO)^[2]:
1 Preparation of 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). Once prepared, store the solution in a sealed container and protect from light.
2 Preparation of ORO working solution
1. Dilute the ORO stock solution to 60% isopropanol with water (3:2 volume ratio).
2 Prior to use, filter the working solution using a 0.2 μm cellulose acetate sterile syringe filter.
Note: For optimal solution quality, it is recommended to prepare the ORO working solution one day in advance and mix overnight. If the solution is to be used the same day, mix the diluted solution on a shaker for at least 2 hours. Wrap the centrifuge tube with parafilm before shaking to prevent leakage.
3 Prepare the sample
1). Grow nematodes in Nematode Growth Medium (NGM) inoculated with OP50 E. coli (late log phase) at 20°C until the target developmental stage.
2). Add 1 mL of PBST solution (containing 0.01% Triton X-100) to the culture dish and gently shake to remove the nematodes from the medium. Tilt the culture dish and rinse with 1 mL PBST, and transfer the nematode suspension to a 1.5 mL centrifuge tube.
3). Centrifuge at 560 × g for 1 minute, discard the supernatant, and keep the pellet. Repeat the wash with 1 mL PBST three times, and finally discard the supernatant and keep only 100 μL.
4). Add 600 μL of 40% isopropanol to the nematode pellet and incubate with shaking at room temperature for 3 minutes.
5). Centrifuge at 560 × g for 30 seconds and discard all but 100 μL of the supernatant, being careful not to disturb the nematode pellet.
Note: If performing high-throughput staining, increase the volume of PBST. Do not wash the nematodes in PBST for more than 15 minutes. If bacteria are still in the supernatant, additional washes are required. Incubate in 40% isopropanol using a shaker or oscillator to ensure that the sample is thoroughly mixed.
4 Staining Liposomes
1). Add 600 μL of ORO working solution to each sample and invert the tube three times to thoroughly mix the nematodes and staining solution.
2). Incubate at room temperature at 30 rpm for 2 hours.
3). Centrifuge at 560 × g for 1 minute and discard all but 100 μL of the supernatant.
4). Resuspend the sample in 600 μL PBST and rotate at 30 rpm for 30 minutes to remove excess staining solution.
5). Centrifuge at 560 × g for 1 minute and discard the supernatant except for 50 μL.
6). To better distinguish the nuclei of the germ cells and intestinal cells of the nematodes, add 1 μL DAPI to each 1 mL of ORO working solution and prepare slides for imaging after 2 hours of staining. The nuclei of intestinal cells are larger, and the gonads can be identified by the developing germ cells.
Note: After ORO staining, the nematodes may adhere to the wall of the centrifuge tube and no obvious pellet may form. If this happens, centrifuge again or let it stand for at least 10 minutes to allow the nematodes to settle.
5 Preparation
1). Resuspend the nematode pellet in the remaining supernatant and mix thoroughly.
2). Place 5 μL of the nematode suspension on the slide and carefully cover with a coverslip to avoid air bubbles.
3). Seal the edges of the coverslip with nail polish before imaging.
Note: The nematodes may be stiff and difficult to pipette after fixation and staining. It is recommended to cut off the tip of the pipette tip to widen the aperture. If imaging is not possible immediately, the slides can be stored in a centrifuge tube rack at 4°C for up to 24 hours.
6 Imaging
1). Photograph ORO-stained nematodes using a microscope camera capable of color imaging.
2). Use a 5X objective to capture multiple nematodes, and switch to a 10X objective to observe individual nematode details.
3). Export images in TIF format to avoid data loss due to compression.
Note: If using ORO+DAPI staining, switch to fluorescence imaging mode.
7 Analysis of Results
1). Import the images into Image J. If the file format is not recognized, you can read it 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 “Brightness/Contrast” to enhance the visibility of lipid droplets.
4). Categorize the images according to the accumulation of lipids throughout the body or in specific tissues.
5). To evaluate lipid localization and identify nonspecific artifacts, select “Stack to RGB” in the “Color” function in the image menu to display the RGB channel signals separately.

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]