Tcr vα7.2 clone 3c10

Five mm thick cut frozen liver sections were thawed and fixed in ice cold 4% paraformaldehyde for 20 min on ice followed by one wash with Tris Buffer Saline (TBS). Sections were incubated with Image-iT® FX signal enhancer for 30 min at RT and washed once in TBS, then blocked with Background Buster (Innovex Biosciences) for 15 min at RT. Sections were then incubated with the primary antibodies targeting TCR Vα7.2 (clone 3C10, Biolegend) and IL-18Rα (clone AF840, R&D Systems), or IgG1 (Biolegend) and Goat (R&D Systems) isotype control antibodies for 1 h at RT. The primary antibodies were detected with the corresponding donkey anti-mouse and donkey anti-goat secondary antibodies conjugated to Alexa 555 or 647 (all Invitrogen). DAPI was included at 0.0005% w/v with secondary antibody. Following staining background autofluorescence was quenched using TrueBlack® Lipofuscin Autofluorescence Quencher (Biotium) for 5 min followed by three brief washes in TBS, then washed once for 10 min in TBS. The slides were mounted using Prolong Diamond (Invitrogen). Sections were visualized with a Nikon Ti-E spinning-disk confocal microscope. Images were acquired at RT using a 40× Nikon air objective, and Andor DU-897 EM-CCD camera (512×512 pixels. Pixel size 16 μm). Images were generated using Cytosketch (CytoCode, Auckland, New Zealand).