Plan apo 63 1.40 na oil immersion objective

Immunofluorescence microscopy was performed as described previously (33 (link)). In brief, cells cultured on coverslips were fixed with 3% formalin in PBS for 10 min at RT, treated with 0.2% Triton X-100 in PBS for 5 min, and then washed with PBS. Blocking was done by incubating the fixed cells with 5% BSA in PBS for 30 min at RT. After the antibodies were diluted with the blocking solution, the cells were incubated at RT for 1 h with the primary antibody and for 1 h with the secondary antibody. For actin staining, Alexa Fluor 488 phalloidin (Life Technologies) was added to the secondary antibody. For imaging of cholesterol-rich domains, cells were cultured on a glass-bottom dish and incubated with recombinant RFP-tagged D4 for 30 min at 37 °C. After washing with HBSS thrice, cells were observed at 37 °C. Both immunofluorescence and live imaging samples were observed with a confocal microscope (LSM700; Carl Zeiss MicroImaging) equipped with a Plan-APO (63/1.40 NA oil-immersion) objective with appropriate binning of pixels and exposure time. The images were analyzed with ZEN 2012 (Carl Zeiss MicroImaging).