Multimodal Imaging of Cultured Cells

For brightfield and epifluorescence, cultured cells were imaged by REVOLVE4 microscope (ECHO) with a 10× objective. For confocal microscopy, samples in eight-well chamber slides were fixed in 4% paraformaldehyde for 10 min at room temperature and stained as previously described (74 (link)). Cells were permeabilized and stained with antibodies against DAPI (P36962, Thermo Fisher), LAMP1 (9091S, Cell Signaling), LBPA (MABT837, Sigma), and Rab4 (ab13252, Abcam) at room temperature for 1 h. For filipin staining, cells were fixed and incubated with 50 μg/mL filipin (SAE0087, Sigma-Aldrich) at 37 °C for 30 min. Stained cells were washed with PBS, whole-mounted with Antifade Mountant, and imaged with a Zeiss LSM880 Confocal Microscope at the Molecular Microbiology imaging core facility at Washington University in St. Louis. Images were visualized by Volocity v6.3 and quantification was determined by ImageJ (NIH).