Alexafluor 647 phalloidin

Cells were grown to approximately 80% confluence on coverslips; they were washed with phosphate-buffered saline (PBS), fixed with 4% PFA/PBS for 20 min at room temperature, washed three-times, permeabilized with 0.1% Triton X-100/PBS for 5 min at room temperature. After washing with PBS, nonspecific binding of the antibodies was blocked with 10% FBS/PBS for 30 min at room temperature. They were then stained for 1 h at room temperature with the rabbit anti-FLAG antibody diluted in 5% FBS/PBS, followed by an anti-rabbit AlexaFluor^® 488 (ThermoFisher Scientific) for one hour at room temperature. In the case of HBcAg detection, an AlexaFluor^® 594 conjugated secondary antibody was used for visualization. For specific staining of the ER and the Golgi, AlexaFluor^® 594 Concanavalin A and AlexaFluor^® 647 Lectin HPA (Helix pomatia agglutinin; ThermoFisher Scientific) antibodies were used respectively to stain the cells for a further hour at room temperature. For specific staining of actin and Tubulin, AlexaFluor^® 647 Phalloidin and a mouse a-Tubulin (Sigma-Aldrich) were used, respectively. For Tubulin, a secondary anti-mouse AlexaFluor^® 594 antibody was used for visualization purposes. Images were taken on an Axiovert 135 TV (Zeiss) fluorescent microscope or an EVOS FL Auto Cell Imaging System (ThermoFisher Scientific), as well as a confocal microscope (LSM Pascal, Zeiss).

For confocal microscopy, cells were fixed with 4% PFA for 30 min at 25 °C and then permeabilizated with 0.1% Triton X-100 for 1 min. The samples were stained with Alexa Fluor 647-phalloidin (Sigma- Aldrich, St. Louis, MO) to visualize the actin cytoskeleton. All samples were mounted on glass slides using ProLong (Molecular Probes Life technologies). Images were acquired using a LSM700 confocal microscope (Zeiss, Germany) equipped with a 63X oil immersion objective and 633 nm laser line. Confocal images were processed using ImageJ. The SEM imaging was carried out as reported elsewhere^{46 (link)}.