Aqua poly mount

Cells infected with FITC (Sigma-Aldrich, F7250)-labelled GAS strains were washed with PBS, fixed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 5 min in 0.2% Triton X-100 in PBS and blocked using 5% BSA for 1 h at room temperature. Then, cells were stained with indicated primary antibodies at 4 °C for overnight, followed by incubation with fluorescent-conjugated secondary antibodies (Jackson ImmunoResearch). Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole) (Cell Signaling Technology, #4083). Slides were mounted using Aqua-Poly/Mount (Dako). Images were captured using a laser scanning confocal microscope (Olympus SpinSR10 Confocal System) with OLYMPUS cellSens Dimension. Z-stack images (optical slice 0.5 μm, 12 slices per 5.5-μm stack) were captured using a Zeiss 880 laser scanning confocal microscope (Zeiss LSM880) at 63× magnification and analyzed using Zeiss Zen software Blue edition.

Cells grown on coverslips were fixed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 5 min in 0.1% Triton X-100 in PBS and blocked using 5% BSA for 1 hr. Then, cells were stained with the indicated primary antibodies followed by incubation with fluorescent-conjugated secondary antibodies (Jackson ImmunoResearch). Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole) (Sigma-Aldrich). Slides were mounted using Aqua-Poly/Mount (Dako). Images were captured using a laser scanning confocal microscope (Olympus Fluoview FV1000 Confocal System) with a 63× water immersion objective and Olympus Fluoview software (Olympus). All confocal images are representative of three independent experiments.