Alexa fluor 488 af 488 conjugated anti mouse antibody

SK-N-SH cells transfected with siRNAs were infected with JEV at an MOI of 1.0 and incubated for 2 h at 37 °C. At 48 h post-infection, the cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 at RT for 10 min. Then, the cells were blocked in 5% bovine serum albumin (BSA) and incubated with an anti-JEV E mouse monoclonal antibody (a gift from The Fourth Military Medical University, Xi’an, China) at room temperature for 2 h. After being washed with phosphate-buffered saline (PBS) three times, the cells were stained with an Alexa Fluor 488 (AF 488)-conjugated anti-mouse antibody (Invitrogen) for 1 h. Nuclei were stained with 4′, 6′-diamidino-2-phenylindole (DAPI, Roche, UK) for 10 min.

Cells seeded on coverslips were incubated with virus at 4 °C for 1 h, and then virus entry was initiated by moving the cells to 37 °C. At the indicated time points, the cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 at RT. The cells were then blocked in 5% BSA and incubated with an Alexa Fluor 488 (AF 488)-conjugated anti-mouse antibody (Invitrogen) and Alexa Fluor 555 (AF 555)-conjugated anti-rabbit antibody (Invitrogen) for 2 h. After three washes with PBS, the cells were incubated with fluorochrome-conjugated secondary antibodies for 1 h. The cells were washed again three times with PBS, and the nuclei were stained with 4′,6-diamidino-2-phenyl-indole (DAPI, Roche) for 10 min. Images were obtained using a confocal laser scanning microscope (Zeiss LSM710 Meta; Carl Zeiss). The fluorescence intensity of the images was processed and quantified using ZEN Light Edition software (Carl Zeiss).