Eclipse e80i fluorescence microscope

Cells were seeded onto sterile, round glass coverslips. After reaching 80% confluence, cells were fixed in 4% paraformaldehyde for 15 min and gently washed with phosphate-buffered saline (PBS). Next, permeabilization with 0.2% Triton X-100 in PBS for 15 min at room temperature was performed. Blocking of non-specific parameters was completed in PBS supplemented with 0.1% Triton and 5% goat serum for 2 h, at room temperature. Primary antibody against PLIN5 (ProGen, #GP31, guinea pig) was diluted 1:200 in PBS and incubated overnight (4 °C, no shaking). For visualization, goat anti-rabbit cross-adsorbed Alexa Fluor 488 was applied at a dilution of 1:1000 in PBS and was incubated for 1 h at room temperature. Slides were subsequently mounted with anti-fade Fluoroshield Mounting Medium with DAPI (#104135, Abcam, Berlin, Germany) and were dried in the dark. The slides were stored at 4 °C until observation. Images were taken using Nikon Eclipse E80i fluorescence microscope and NIS-Elements Vis software version 3.22.01 (Nikon, Tokyo, Japan).

A Rhodamine-Phalloidin-conjugate (#R415, Thermo Fisher Scientific) was used to stain the microfilaments in PAV-1 cells according to a previously established protocol [19 (link),35 (link)]. The nuclei were counterstained by incubation in a 4′,6-diamidino-2-phenylindole (DAPI) solution (#D1306, Thermo Fisher Scientific), and the glass coverslips were mounted with PermaFluor aqueous mounting medium (#TA-030-FM, Thermo Fisher Scientific). Images of stained cells were captured using a Nikon Eclipse E80i fluorescence microscope (Nikon Europe, Düsseldorf, Germany) equipped with the NIS-Elements Vis software (Nikon Europe, version 3.22.01).