Goat anti mouse coupled to alexafluor 633

A549 cells grown on Lab-Tek II chamber slides (ThermoScientific) were fixed with 4% paraformaldehyde in PBS for 30 min. After washing in PBS, cells were permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 15 min. Mouse monoclonal anti-nucleoprotein (17C2, VirPath laboratory)79 (link), mouse monoclonal anti-nucleoprotein clone 3/1 (MAb 3/1, kind gift from R. Webster), anti-B23 (Sigma), rabbit polyclonal anti-nucleolin (#134, kindly provided by P. Bouvet) and anti-fibrillarin (Abcam, Ab5821) antibodies were used as primary antibodies in PBS-T. After incubation for 1 h, the cells were washed in PBS-T and then incubated with goat anti-mouse coupled to AlexaFluor 633 and/or goat anti-rabbit coupled to AlexaFluor 488 (Molecular Probes, Invitrogen) for 30 min, at concentrations recommended by the suppliers. Nuclei were counterstained with DNA-binding fluorochrome 4,6-diamidino-2-phenylindole (DAPI, Invitrogen). After staining, the coverslips were mounted with Fluoromount G (Cliniscience) and analyzed using a confocal laser scanning microscope (Leica). Each observation was performed on at least 20 individual cells from 5 different large fields of view. For all observations, each representative pattern was reported.

A549 cells grown, and Mock or IAV-infected on Lab-Tek II chamber slides (ThermoScientific) were fixed with 4% paraformaldehyde in PBS for 30 min. After washing in PBS, cells were permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 15 min. Mouse monoclonal anti-Mdm2 antibody (SMP14, sc-965, Santa Cruz Biotechnology), and a rabbit polyclonal anti-NS1 (Kind gift of Dr Juan Ortin, CSIC, Spain) were used as primary antibodies in PBS-T. After incubation for 1 h, the cells were washed in PBS-T and then incubated with goat anti-mouse coupled to AlexaFluor 633 and/or goat anti-rabbit coupled to AlexaFluor 488 (Molecular Probes, Invitrogen) for 30 min, at concentrations recommended by the suppliers. Nuclei were counterstained with DNA-binding fluorochrome 4,6-diamidino-2-phenylindole (DAPI, Invitrogen). After staining, coverslips were mounted with Fluoromount G (Cliniscience) and analyzed using a confocal laser scanning microscope (SP5 Leica). The relative mean nuclear intensity of NS1 and Mdm2 stainings was measured with ImageJ (version 1.51 h - http://imagej.nih.gov/ij), using DAPI staining to define nuclear areas for measurements. Data collected for NS1 and Mdm2 were subject of a correlation analysis using Graphpad Prism software (La Jolla, California, USA).