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1. Oil Red O Staining Preparation
1.1 Oil Red O solution stock: Dissolve 0.5 g Oil Red O in 200 ml isopropyl alcohol at 56 ℃ for 1 h. Cool down before further use. Store at room temperature.
1.2 Oil Red O working solution: Make fresh the day of use. Mix four parts distilled water with six parts of stock solution. Let solution rest for at least 15 min. Precipitate will form. Filter solution through a filter paper, for example, 3MM Chr Whatman paper. Keep at room temperature until use.
1.3 1×PBS, pH 7.4.
1.4 60% (v/v) isopropyl alcohol in distilled water.
2. Fixing cells
2.1 Wash cells very carefully with PBS as they are easy to detach from the plate.
2.2 Add 1 ml of 4% PFA per well and incubate for 20 min at room temperature on a level surface with no shaking or tilting.
2.3 Discard the PFA in a hazardous waste chamber and wash cells twice for 5 min in PBS.
3. Mounting of cover slips
3.1 Add a 40 ml drop of mounting solution in the middle of a glass slide.
3.2 Remove the cover slip from the six-well plate using tweezers and dry the edges by blotting with tissue paper. Then place the cover slip on the mounting solution (side with cells facing down) taking care not to trap bubbles.
3.3 Incubate 2 h at room temperature in the dark to let the mounting solution solidify.
3.4 Wipe off any precipitate on the cover slip with a moist tissue paper.
3.5 Keep the slides in the dark at 4 ℃.
4. Oil Red O staining
4.1 Fix cells and remove PBS (see Section 2).
4.2 Wash cells with 60% isopropyl alcohol.
4.3 Add 1.5 ml of Oil Red O working solution per well and incubate for 15–30 min. Observe under microscope until cells are properly stained. Make sure to stop the staining when/if precipitate starts to form. Wash cells with 60% isopropyl alcohol.
4.4 Wash cells with 1 ml PBS and leave in PBS until further use.
4.5 Mount cover slips on glass slides or use the stained cells for quantification by Oil Red O extraction. The Oil Red O-stained cells can be photographed directly, or higher magnification images of cells on the mounted cover slips can be obtained using any microscope equipped with an RGB camera.
This protocol only provides a guideline, and should be modified according to your specific needs.
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