Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

de Vries, Erik; Tscherne, Donna M.; Wienholts, Marleen J.; Cobos-Jiménez, Viviana; Scholte, Florine; García-Sastre, Adolfo; Rottier, Peter J. M.; de Haan, Cornelis A. M.

Cells were fixed with 3.7% paraformaldehyde (PFA) in PBS and subsequently permeabilized with 0.1% Triton-X-100 in PBS. After blocking with normal goat serum IAV-infected cells were incubated for 1 h with a monoclonal antibody directed against the nucleoprotein (NP) (HB-65; kindly provided by Dr. Ben Peeters). After washing, the cells were incubated with a 1:400 dilution of Alexa Fluor 488-or 568-labeled goat anti-mouse IgG (Molecular Probes) secondary antibody for 1 h. Nuclei were subsequently stained with TOPRO-3 and after three washing steps, the coverslips were mounted in FluorSave (Calbiochem). Actin was stained using phalloidin labeled with Alexa Fluor 633. The immunofluorescence staining was analyzed using a confocal laser-scanning microscope (Leica TCS SP2). FITC, GFP or Alexa Fluor 488 were excited at 488 nm, Alexa Fluor 568 at 568 nm, and TOPRO-3 at 633 nm.