Oil red o staining solution

For oil red O staining, the ICP-1 cells were washed with PBS (Gbico, New York, United States) and then fixed with 4% paraformaldehyde solution (Sangon Biotech, Shanghai, China) for 30 min at room temperature. After fixation, the cells were rinsed twice with PBS, and oil red O staining solution (Sangon Biotech, Shanghai, China) was added followed by the incubation of cells for 60 min at room temperature. Then, the staining solution was removed, and the cells were washed three times with PBS. Image analysis was carried out using a DMi8 microscope (Leica, Wetzlar, Germany). The oil red O dye in stained cells was extracted with isopropanol solution and the absorption value was measured at 510 nm using a microreader (Bio-Tek, Vermont, United States).

The oil red O staining method was used to detect lipid droplets in HC11 cells. Cells were washed with PBS (Sigma Aldrich, St. Louis, MO, United States) for 3 times, then added with 4% paraformaldehyde and fixed at room temperature for 40 min. Subsequently, HC11 cells were washed twice with PBS, then added oil red O staining solution (Sangon Bio, Shanghai, China), and incubated at room temperature for 3 h. Discard the staining solution and rinse HC11 cells with 65% isopropanol once. Then HC11 cells were washed with PBS 5 times and observed under an inverted microscope (400×). In this study, Triglyceride Assay Kit (Jiancheng, Nanjing, China) was used to detect the content of TAG.

To observe the production of milk fat more intuitively, oil red O staining solution (Sangon Bio, Shanghai) was used to observe the lipid droplets. In this experiment, the oil red dye solution used was diluted with double distilled water in a ratio of 6:4, and then filtered through 45-nm filters for 3 to 5 times before it can be used as a working solution. The cells were seeded into 24-well plates and cultured according to the methods mentioned above. They were harvested 24 h after being treated with the treatment medium for oil red staining. In brief, the cells were rinsed 3 times with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Then, fresh working solution was added to cells (200 μL per well) followed by rinse with PBS twice. After 6 h of incubation at room temperature, cells were rinsed with PBS until there were no visible pellets before being observed and photographed under a microscope.