Axiovert 200 confocal microscope

A549 cells grown on coverslips were transfected with PCAGGS and PCAGGS-SOD1. After 24 h post-transfection, the cells were washed twice with F-12 medium without FBS. The cells were infected with five multiplicity of infection (MOI) of DW. After absorption was performed in an incubator with 5% CO₂ for 1 h at 37 °C, the cells were washed twice. F-12-containing 0.5% FBS was added and incubated for 6 h. The cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. Afterward, the cells were permeabilized in 0.1% to 0.2% Triton X-100 for 10 min, and blocked with 1% BSA for 1 h at room temperature. The cells were incubated in PBS containing mouse anti-NP and rabbit anti-HA antibodies at 4 °C overnight. The cells were then incubated in PBS containing Alexa 488- and 594-conjugated goat anti-mouse or anti-rabbit secondary antibodies. After a final wash, the cells were stained with 4,6-diamidino-2-phenylindole (1 μg/mL in methanol) for 10 min. The fluorescence was visualized under Axiovert 200 confocal microscope (ZEISS, Oberkochen, Germany). Fifty cells expressing PCAGGS or PCAGGS-SOD1 were counted to determine the rate of NP nuclear export.

Immunohistochemistry and immunofluorescence were performed according to previously described methods55 (link)56 (link). After incubation with the primary antibody (Supplemental Table 1) overnight at 4 °C in a humidified chamber, the sections were stained using the avidin-biotinylated-peroxidase ABC kit followed by a 5-min treatment with DAB (SK-4100, Vector Laboratories). Sections were imaged on a Nikon E-1000 microscope (Japan) under bright-field optics and photomicrographed using Easy Image 1 (Bergström Instrument AB, Sweden).
After the sections were incubated with primary antibodies in TBST containing 5% nonfat milk overnight at 4 °C in a humidified chamber, a secondary antibody was applied at room temperature for 1 h. After the sections were washed with TBST, they were re-suspended in mounting medium containing DAPI (4′,6′-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA) and examined under an Axiovert 200 confocal microscope (Zeiss, Jena, Germany) equipped with a laser-scanning confocal imaging LSM 510 META system (Carl Zeiss) and photomicrographed.