Mouse skin tissue histology and immunofluorescence staining.

Skin and infected lesions excised from mice were fixed in 10% buffered formalin and submitted to the Translational Research Institute Histology Core Facility at The University of Queensland for processing and immunofluorescence staining. Briefly, the samples were processed in a Tissue-Tek VIP6 tissue processor and embedded with a Leica embedding station, which allows tissues to be oriented with the correct direction and then forms them into paraffin blocks. The tissue blocks were then sectioned at a 4-μm thickness and stained with hematoxylin and eosin (H&E) using Tissue-Tek Prisma and Glas for automated staining and coverslip addition. The histology slides were viewed and photographed under a Zeiss Axioplan 2 light microscope with a high-resolution Axiocam digital camera. We used the discovery ultra Ventana instrument (Roche) to perform immunofluorescence staining. The primary antibodies were used at a concentration of 1:100 to stain neutrophils (mouse anti-CD63 antibody; Abcam) and calprotectin (mouse anti-S100A9 antibody; Abcam) and a concentration of 1:300 to stain GAS (rabbit anti-group A carbohydrate antibody; Abnova). The secondary antibody was anti-mouse HQ antibody, supplied by Roche Ventana, followed by anti-HQ horseradish peroxidase (HRP) antibody, for 16 min each. The slides were stained with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin (Hx).