Oil red o stain

Mouse monoclonal anti-human CD61-FITC (Dako, Denmark), mouse monoclonal anti-human CXCR4-PE (BioLegend, USA), mouse monoclonal anti-human/mouse CXCR7-PE (BioLegend, USA), mouse monoclonal anti-human SDF-1-PerCP (Novus Biologicals, USA), mouse monoclonal anti-human CD14-PE (Beckman Coulter, USA), mouse monoclonal anti-human CD11b-PE (Dako, Denmark), mouse monoclonal anti-human CD11c-FITC (Dako, Denmark), mouse monoclonal anti-human CD86-PerCP (Abcam, USA), and mouse monoclonal anti-human CD163-FITC (R&D Systems, UK) were used in our study. Ficoll-Hypaque was from Lymphodex (Inno-Train, Germany), Taq DNA Polymerase 2x Master Mix RED and Real Q Plus Master Mix Green Low ROX were procured from Ampliqon (Copenhagen, Denmark), the RevertAid First Strand cDNA Synthesis Kit was from Thermo Fisher Scientific (USA), and trypan blue, Oil Red O (ORO) stain, and TRIzol were purchased from Sigma-Aldrich (USA).

Tissues were stained with hematoxylin and eosin using a standard protocol. Liver tissues were stained using Oil Red O (ORO) stain (Sigma-Aldrich, O0625). An initial stock of 0.5% ORO in 2-propanol was diluted to 0.3% ORO in 60% isopropanol and filtered into a Coplin jar for staining. PFA-fixed sections were rinsed once with 60% isopropanol then immersed in 0.3% ORO in 60% isopropanol for 12 min. Sections were rinsed with 60% isopropanol for 3-4 min to remove excess ORO. Nuclei were stained with hematoxylin solution (5 dips) and rinsed with distilled H₂O for 3 min. Sections were mounted with 100 µl ImmunoHistoMount solution (Sigma, I1161). Staining was viewed and imaged using a Nikon 80i microscope equipped with a Nikon DS-Fi1 camera.

HepG2 cell cultures (1.2 × 105 cells/well) were incubated at 37 °C with DMEM (K-), DMSO (K+), PA/OA, PA/OA + 80 μM dynamin blocker (Dynasore, Abcam, UK) and/or PA/OA and 9 μM FATP2 blocker (CB16.2). The steatotic cell cultures were treated with Dynasore and/or CB16.2 for 1 h; the FATP2 blocker was kindly provided by Prof. Concetta C. DiRusso, University of Nebraska [23 (link)]. For the dynamin assay, culture medium without serum was used as serum inhibits the effect of the Dynasore while studies with CB16.2 to block FATP2 were conducted in the presence of serum.
Post treatment, cells were harvested, washed three times with PBS and fixed with 4% formaldehyde. After incubation at room temperature for 20 min, the cells were washed with deionized water and treated with 60% isopropanol for 3–5 min and finally suspended in oil red O (ORO) stain (Sigma Aldrich, Germany) and incubated at room temperature for 10 min. After staining, the cells were washed several times with deionized water to remove excess stain and re-suspended in 100 μl running tap water. The ORO stain was imaged with Texas Red filter (excitation562/emission624) by phase contrast fluorescence and light microscopy using the Nikon TiE microscope. Furthermore, quantification of the intracellular lipid was done as described before,  and statistical testing involved a t-test and was considered significant at p < 0.05.