Oil red o

The accumulation of fatty acids was detected by staining Huh-7 cells with Oil Red O (catalog #154-02072) from Wako (Richmond, VA, USA). Huh-7 cells were cultured on coverslips in 12-well plates and treated as described above. Cells were washed with phosphate buffered saline (PBS) and fixed in 10% formalin. After washing with PBS, Oil Red O was added. Coverslips containing the cells were transferred onto the slides and mounted using aqueous mounting media. Cells were observed under brightfield (EVOS FL Cell Imaging System, Thermo Fisher Scientific), fluorescence (using FITC and DAPI Fluo channels) and phase contrast (3D Cell Explorer, NanoLive) microscopy and photographs were taken.

Accumulation of fatty acids was detected by staining with Oil Red O (Wako, 154–02072). Huh7 cells were incubated in the 6-well plates overnight and treated with FFA. Dishes with control cells (cells without FFA treatment) and FFA-treated cells were washed with phosphate buffered saline (PBS) and fixed in 10% formalin. After washing by PBS, Oil Red O was added.

The liver was resected at various time points, fixed with 4% buffered paraformaldehyde solution, embedded in paraffin, and sectioned into 5-µm thickness. Oil red O (Wako Pure Chemical Industries) staining was performed to confirm fatty deposition. Sirius red (saturated picric acid containing 0.1% Direct Red 80 and 0.1% Fast Green FCF; Sigma-Aldrich, St. Louis, MO, USA) staining was done to visualize collagen deposition. Stained fibrotic areas were measured as percentage area in a representative ×100 high-power field in each mouse using Image J software. For the immunostainings the sections were incubated overnight with the primary antibodies at 4°C, after which the secondary antibodies were added. Kupffer cells or macrophages were stained with rat anti-F4/80 monoclonal antibody (Abcam). TNF-α staining was performed with anti-TNF-α goat polyclonal antibodies (R&D systems, Minneapolis, MN, USA). Activated myofibroblasts were stained with anti-α-smooth muscle actin (SMA) rabbit polyclonal antibody (Abcam). Negative controls were prepared with isotype IgG.

The whole length of the isolated aorta was placed in 4% paraformaldehyde and stored at 4 °C. After carefully and completely removing minor branching arteries and fat tissues from its exterior, the aorta was opened longitudinally and stained using Oil Red O (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to identify atheromas. Lesion areas were measured and quantified using the ImageJ software (version 1.52; https://imagej.nih.gov/ij/). All morphological analyses were performed in a blinded manner.