Fluoview 2

293 T cells were seeded into 0.8 cm²-well chamber slides so that the cells were 60% confluent at the time of transfection. Cells were transfected with 0.3 μg of each plasmid using Lipofectamine^TM 2000, as described by the manufacturer, for 24 hours. Cells were fixed and permeabilized in 4.0% paraformaldehyde-PBS + 0.6% Triton^®-X-100 (Sigma-Aldrich, Oakville, Ontario, Canada) and blocked in PBS supplemented with 1% Bovine Serum Albumin and 0.6% Triton^®-X-100. A monoclonal antibody against NiV P generated in house was employed for the detection of cell-expressed NiV P, followed by the secondary antibody, goat anti-mouse-AlexaFluor^® 568 (Life Technologies, Burlington, ON, Canada), and finally, a monoclonal antibody generated in house, mouse anti-NiV N-FITC, against NiV N was added. An Olympus IX70 confocal microscope and Fluoview 2.1 software were used for acquisition of images. Cells were visualized at 60X magnification. Controls to ensure that cross-reactivity between antibodies or cells was not occurring were carried out for each experiment.

The nucleic acids of the whole cells were visualized using the specific SYBR Green fluorescent dye (1:100 dilution), on samples not handled further, under an Olympus FluoView FV1000 confocal laser scanning microscope, and the 488-nm excitation laser line with emission signal being collected at 510–530 nm. Images were analyzed with the FluoView 2.1 software (Olympus). FESEM images were acquired using FEI Teneo (Thermo Fisher, MA, USA). To this end, samples were prepared as reported in Addesso et al. [17 (link)]. In particular, they were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4 °C for 2 h and washed thrice in cacodylate buffer. Subsequently, they were treated with 1% osmium tetroxide for 1 h at 4 °C and dehydrated by subsequent dilution series in ethanol and acetone finishing with 100% acetone before drying. The samples were dried in a EM CPD 300 (Leica Microsystem, Wetzlar, Germany) critical point drying device at 34.5 °C. Finally, samples were mounted on SEM stubs and sputter-coated with gold (5–10 nm).