Immunohistochemical Analysis of cNT Sections

At the previously identified endpoint, cNTs were collected as described [37 (link)] and tissues were fixed in periodate-lysine-paraformaldehyde (PLP) and prepared for sectioning as previously [73 (link)]. Following fixation, cNT were passed through sucrose gradients and frozen in OCT (TissueTek) media. Serial sections (7 μm) were cut using a cryostat. Prior to staining, all slide-mounted tissue sections were blocked with PBS containing 1% BSA, 0.1% Tween-20, and 10% rat serum. Sections were stained with the following antibodies: anti-Streptococcus pyogenes Group A Carbohydrate (Abcam, ab9191), anti-B220 (Biolegend, RA3-6B2), anti-CD3 (Biolegend, 17A2), and anti-Ly6G (Biolegend, 1A8). After staining, sections were mounted with ProLong Gold Antifade Reagent (Invitrogen). Tiled images of whole cNT sections were collected using a DM5500B fluorescence microscope (Leica) at 10× and 20×.