Hp cl apo 63 1.40 oil cs2 oil immersion objective

Confocal laser scanning microscopy was conducted using a Leica TCS SP8 system (Leica Microsystems, Wetzlar, Germany). Cells were seeded in ibitreat dishes (ibidi, Munich, Germany) and grown for 72 h. For immunofluorescence staining, cells were fixed using 4% PBS-buffered formaldehyde and subsequently stained with primary and secondary antibodies using standard protocols. Counterstaining of filamentous actin and nuclei was carried out using phalloidin-TRITC and DAPI, respectively. Excitation of fluorophores was achieved using integrated lasers emitting at 405 nm (for DAPI), 488 nm (for secondary antibodies labeled with Alexa488), 552 nm (for phalloidin-TRITC) and 638 nm (for secondary antibodies labeled with Alexa647). For AO live cell imaging, excitation of the dye was performed using the 488 nm laser. Neutral compartments of the cell were detected by collecting emitted light at 493–547 nm (designated as “AO neutral”), whereas acidic compartments were detected by collecting emitted light at 575–739 nm (designated as “AO acidic”), respectively. Fluorescence was detected using a HP CL APO 63 × /1.40 OIL CS2 oil immersion objective (Leica Microsystems) and a pinhole setting of 1 Airy unit.