2.3. Immunofluorescence Microscopy

HNC cells were plated at a density of 30,000 cells/mL on sterile round glass coverslips in a 12-well dish. The following day, the medium was replaced with a fresh serum-free medium, with or without 30 µg oxLDL. After 48 h of exposure, cell monolayers were fixed with 4% paraformaldehyde in DPBS. Before the application of antibodies, the cell monolayers were rinsed several times with PBS (0.04 M Na₂HPO₄, 0.01 M KH₂PO₄, 0.12 M NaCl, pH 7.2) containing 0.1% Triton X-100 (the same detergent-containing buffer was used for subsequent incubations and rinsing steps). Before exposure to the primary antibodies, the cells were preincubated for 20 min in PBS containing 0.05% casein to prevent non-specific adsorption of immunoglobulins. Cells were exposed overnight to the primary antibody (either anti-CD36 (Merk Sigma, Darmstadt, Germany) or anti-Lox-1 (ThermoFisher Scientific, Waltham, MA, USA)), which had been diluted at 1:200 and 1:1000, respectively, in PBS containing 0.05% casein. On the following day, the cells were exposed to an anti-rabbit IgG antibody coupled with Alexa 594 for 30 min (ThermoFisher Scientific, Waltham, MA, USA). After the final rinses in PBS, the coverslips were mounted onto glass slides using a commercial anti-fading medium (Vectashield, Vector Laboratories, Burlingame, CA, USA). Confocal microscopy observations were carried out using an Olympus FV1000D laser scanning inverted microscope equipped with a red laser diode (LD559) (Olympus, Tokyo, Japan).