Alexa fluor 488 labelled anti mouse secondary antibody

HEK293T cells stably expressing the EBNA1-FLAG-HA protein were seeded in 24 well plates at a density of 5x10⁴ cells/well. The cells were grown for 24 h and washed twice with PBS and fixed with 4% paraformaldehyde and 4% sucrose for 20 min at 22°C. Cells were permeabilized with 0.15% triton X-100 in PBS for 5 min at 22°C and blocked in 10% normal goat serum (Wisent). The EBNA1 protein was stained with primary mouse anti-HA antibody (Abcam) for 4 h at 22°C. Cells were washed and incubated in the dark for 1 h at 22°C with an Alexa Fluor 488-labelled anti-mouse secondary antibody (Abcam). Nuclei were stained with Hoechst (1 μg/ml) for 15 min at 22°C. Cover glasses were affixed on slides with SlowFade mounting medium (Invitrogen S36937). Epifluorescence microscopy was conducted using a Nikon Eclipse TE2000-E visible/epifluorescence inverted microscope using bandpass filters for Hoechst and Alexa Fluor 488.

Vero E6 cells were transfected with 2 μg of the pcDNA3-HA plasmid DNAs encoding the PUUV NSs protein derivatives using Lipofectamine 3000 (Thermo Fisher). For transfection, 2 × 10⁵ cells were seeded simultaneously with transfection mix in a 6-well plate. Forty-eight hours after transfection, the cells were fixed with 4% paraformaldehyde and stained for immunofluorescence analysis using an HA-specific mouse monoclonal antibody (HA-probe antibody (F-7): sc-7392, Santa Cruz Biotechnologies) and an Alexa Fluor 488–labelled anti-mouse secondary antibody (Abcam). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). For confocal microscopy, cells were seeded on glass cover slips, which were then mounted with Ibidi mounting medium (Ibidi).