Spinsr10 confocal system

Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, cut into sections, and placed on adhesion microscope slides. Sections were subjected to H&E and immunohistochemical staining according to standard procedures. Stained tissue sections were imaged by light microscopy (Olympus BX53) with CellSens software. Neutrophils infiltrated into the epidermis and dermis were enumerated in a blinded fashion according to neutrophils-specific anti-myeloperoxidase immunohistochistry staining (ten images per mouse, five mice per group) using Image J software. For fluorescence microscopy, skin tissues from Gsdma1^−/− mice infected with FITC-labelled GAS were embedded with Tissue Tek OCT compound (Sakura Finetech) and snap-frozen in liquid nitrogen. Tissue sections were then stained with the indicated primary antibodies and DAPI using standard methods. Images were captured using a laser scanning confocal microscope (Olympus SpinSR10 Confocal System) with OLYMPUS cellSens Dimension.

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.