Fluoview 1000 imaging software

Huh-7.5-overexpressing cells were seeded on glass coverslips, fixed with 3% PFA at room temperature for 10 min on the following day, and washed twice with PBS. Subsequently, cells were treated with PBS containing 0.1% Triton X-100 for 10 min at room temperature to permeabilize. After washing twice with PBS, coverslips were incubated in a 5% solution of goat serum in PBS for 1 h at room temperature to block potential nonspecific antibody binding. For CD302 detection, human CD302/CLEC13A mouse monoclonal antibody (R&D Systems) was diluted to 5 μg/mL and incubated at room temperature for 1 h or overnight at 4°C. Afterward, coverslips were washed three times with PBS, followed by an incubation with anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific) in a 1:1,000 dilution at room temperature in the dark for 1 h. After washing again twice with PBS, the nuclei were stained using a 0.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) solution in water. Finally, after one more washing step with water, mounting of the coverslips in ProLong Gold antifade mountant (Thermo Fisher Scientific) was performed, and they were left to dry overnight at room temperature in the dark. Images were taken with an inverted confocal laser-scanning microscope (Olympus FluoView 1000) with FluoView 1000 imaging software (Olympus, Tokyo, Japan).