Hc pl apo 63 1.30 glyc corr cs2 objective

Briefly, cells were fixed with 4% PFA in PBS for 15 min at room temperature (RT), washed three times with 0.2% bovine serum albumin (BSA) in Dulbecco’s PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were blocked in 1× Dulbecco’s PBS supplemented with 0.2% BSA. Both primary and secondary antibodies were applied onto cells and incubated at RT for 1 h. The following antibodies were used for immunofluorescence staining: mouse monoclonal FHOD1 (Santa Cruz sc-365437; 1:100), mouse Dia1 (BD biosciences 610848; 1:100), and rabbit monoclonal CAV-1 (CST 3267; 1:100). Alexa-conjugated phalloidin was added together with primary antibody solutions onto cells. All immunofluorescence data were obtained with upright Leica SP8 confocal microscope with HC PL APO 63×/1.30 GLYC CORR CS2 objective. The pixel size was optimized properly to achieve the maximum resolution, which was calculated to be 63.3 nm. The CAV-1 pit size was thus defined as no more than 2 × 2 pixel and the CAV-1-clustered rosette size was defined as no less than 3 × 3 pixel. For detection and measure of the cytoplasmic CAV-1-tagged vesicles, the ‘Spots’ tool of Imaris 9.2 (Bitplane) was used with the configuration defined as 2 μm for estimated XY diameter. The numbers and sizes of spots were calculated subsequently.