Ts100 f fluorescence microscope

Fourteen days after inoculation, A549 cells were washed three times with phosphate-buffered saline (PBS) for 5 min. After washing, cells were fixed with 80% acetone at −20 °C for 10 min and blocked with 1X PBS containing 0.5% BSA for 30 min. Subsequently, cells were rinsed with PBS and stained with mouse-HEV ORF2 antibody (MAB8002) (1:200 dilution; Millipore, Billerica, MA). After overnight incubation, cells were washed with PBS and stained with fluorochrome-conjugated anti-mouse IgG secondary antibody (4408) (1:200 dilution; Cell Signaling, Beverly, MA) at 37 °C for 1 h. Finally, cells were washed and counterstained with 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Fluorescence was examined and images analyzed using an inverted Nikon TS100-F fluorescence microscope (Tokyo, Japan) equipped with a digital camera and Nikon NIS-Elements microscope imaging software.

Cells were fixed with a 1:1 mixture of methanol and acetone for 10 min at −20 °C, washed with 1X PBS, and blocked with 1% BSA at room temperature for 1 h. Subsequently, cells were incubated with primary antibody overnight at 4 °C, followed by secondary antibody at room temperature for 1 h. After staining of cells with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA), fluorescence was examined under a Nikon TS100-F fluorescence microscope (Tokyo, Japan) equipped with a digital camera. Fluorescence images were analyzed using Nikon NIS-Elements microscope imaging software. An antibody against HCV core protein was purchased from Anogen (Mississauga, ON, Canada). Alexa 488-labeled secondary antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA).