880 confocal microscopy

Staining was performed on paraffin-embedded sections from tumor tissues. Briefly, all tissues for paraffin-embedding were resected, rinsed in PBS, and placed in 4% PFA for over 48 h at 4 °C. Immunofluorescence staining was performed by deparaffinization, antigen retrieval, permeabilization, and blocking in 1% bovine serum albumin. All antibodies conjugated with fluorophores were incubated overnight at 4 °C, followed by nuclei counterstained with Prolong® Diamond Antifade Mountant with DAPI (ThermoFisher Scientific). Stained slides were imaged with Zeiss 880 Confocal microscopy (Germany). Three randomly microscopic fields were selected and quantified by Image J software.

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.