Ix73 immunofluorescence microscope

Vero cells with 90% confluence were infected with aMPV/C (MOI = 0.5) in 24-well culture plates. The cells were fixed with precooled and permeabilized using 4% paraformaldehyde (Sigma-Aldrich, 16005), 0.1% Triton X-100 (Sigma-Aldrich, T8787) and in 2% BSA (Beyotime, ST023) in PBS at different indicated time points, and anti-N monoclonal antibody and anti-MAVS rabbit polyclonal antibody were co-incubated with the cells for 2 h at 37 °C. After 3 washes with PBS-Tween-20 (PBST containing 0.05% Tween-20 [Sigma, P1379]), the cells were co-incubated with secondary fluorescein isothiocyanate (FITC)-conjugated antirabbit and Tetramethylrhodamine-6-isothiocyanate (TRITC)-conjugated antimouse antibodies for 2 h at 37 °C. Finally, the cells were washed with PBST and directly observed under an Olympus IX73 immunofluorescence microscope.

The cells were fixed with 4% paraformaldehyde (PFA) for 30 min at 4°C, and then washed in cold PBS for 5 min 3 times. The nuclei were subsequently permeabilized with PBS with 0.5% Triton X‐100 for 30 min. Next, the cells were blocked with 1% BSA in PBS for 1 h. The cells were incubated with primary antibody overnight at 4°C. Primary antibodies used were cleaved CASPASE 3 antibody (9664S, CST), Ki67 antibody (ab15580, Abcam), NESTIN antibody (Santa Cruz, sc‐58813), NR2E1 antibody (Santa Cruz, sc‐377240X), GFAP antibody (Millipore, 5541) and TUJ‐1 (abcam, ab1445). After wash, the cells were incubated with anti‐rabbit secondary antibody conjugated with proper Alexa Fluor label for 1 h at room temperature in darkness. The nuclei were counterstained with DAPI. The cells were imaged with Olympus IX‐73 immunofluorescence microscope.