Uplanapo 100 1.5 oil objective

Immunofluorescence (IF) experiments were performed as previously described (Jiu et al., 2019 (link)). Briefly, cells were fixed with 4% PFA in PBS for 15 min at room temperature (RT), washed three times with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were then blocked in PBS supplemented with 5% BSA. Both primary and secondary antibodies were applied onto cells and incubated at RT for 2 h. Alexa-conjugated phalloidin was added together with secondary antibody solutions onto cells. Alexa 647-wheat germ agglutinin (WGA) (#W32466, Thermo Fisher Scientific) was used to visualize the plasma membrane. All IF data were obtained with Olympus SpinSR10 Ixplore spinning disk confocal microscope with UplanApo 100×/1.5 Oil objective (Olympus Corporation, Tokyo, Japan). The pixel size was optimized properly to achieve the maximum resolution which was calculated to be 65 nm. For detection and measure of the cytoplasmic CAV-1 tagged vesicles, the “Spots” tool of Imaris 9.2 (Bitplane, Zurich, Switzerland) was used with the configuration defined as 2 μm for estimated XY diameter. The numbers and sizes of spots were calculated subsequently. For detection and measure of the lamellipodia width, a plot profile perpendicular to the plasma membrane was made by using imageJ, and the peak zone was defined as the width.