Endocytosis of RSV envelope proteins in Hep-2 cells. Hep-2 cells were infected with RSV at a multiplicity of infection of 2. After 12 h, the cells were incubated with anti-RSV goat antibody at 4°C, rinsed and subsequently incubated at 37°C at different times (0, 30 and 60 min) to allow endocytosis to occur. The assay was performed in the presence of diluting agent dimethyl sulfoxide (DMSO), methyl-beta-cyclodextrin (MBCD) or monodansylcadaverine (MDC). The cells were fixed/permeabilized with ice-cold methanol:acetone and incubated with anti-goat IgG-FITC antibody. Later, unbound antibody was washed away and the cells were finally mounted in VectaShield Propidium Iodide medium and analysed on an Olympus FV1000 confocal microscope. RSV antigens and nuclei appear in green and red, respectively.