Macrophages were seeded onto 6-channel μ-Slide VI 0.4 (ibidi) at 2.63 × 104 cells/cm2 and followed the same cholesterol-loading and treatment procedures for 32 μM LXR or LXR PA epitope equivalent as described in the cholesterol efflux assay. All PAs contained a red fluorescence signal from either TAMRA or AlexaFluor 555-conjugation. After overnight treatment, the cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS, permeabilized with 0.125% Triton-X in PBS for 10 minutes, rinsed with PBS, and blocked with 2% w/v BSA for 1 hour at RT. The samples were then incubated with 5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) and phalloidin (1:1000 dilution, CruzFluor™ 647 conjugate, Santa Cruz Biotechnology) in PBS for 1 hour at RT, rinsed, and stored in PBS at 4 °C until analysis.
Microscopy was performed using Zeiss 880 confocal microscopy at 63× magnification as previously described.[12 ] Video animations of the confocal images were compiled using Imaris software version 9.5.1 (Oxford Instruments). To examine colocalization of PAs to cholesterol, we performed Manders coefficient calculations using the Coloc 2 plugin from Fiji. The nuclei, shown by DAPI staining, were subtracted from the cytoskeleton, shown by phalloidin staining. The resulting area was combined with the cholesterol area for colocalization analysis.
Microscopy was performed using Zeiss 880 confocal microscopy at 63× magnification as previously described.[12 ] Video animations of the confocal images were compiled using Imaris software version 9.5.1 (Oxford Instruments). To examine colocalization of PAs to cholesterol, we performed Manders coefficient calculations using the Coloc 2 plugin from Fiji. The nuclei, shown by DAPI staining, were subtracted from the cytoskeleton, shown by phalloidin staining. The resulting area was combined with the cholesterol area for colocalization analysis.