Protein Detection from Frozen Renal Tissue

Protein detection from renal tissue preserved at −80°C was performed as previously described (20 (link)). Briefly, 50 μg of total protein were resolved in a 4–12% NuPAGE Bis-Tris gradient gel (Invitrogen). Proteins were immobilized onto a nitrocellulose membrane (Invitrogen) and blocked with 10% BSA for 30 min at room temperature. Membrane was incubated with rabbit anti-mouse IL-18 clone H-173 (1:500, Santa Cruz) or anti–tubulin (1:1,000, Cell Signaling Technology) overnight at 4°C. After three washes with PBS-Tween, membrane was probed with secondary IRDye800 goat anti-mouse or IRDye800 goat anti-rabbit antibodies (1:10,000, Li-Cor Biosciences, Lincoln, NE) for 1 h at room temperature. Proteins were detected using the Li-Cor Odyssey Infrared Imaging System following manufacturer’s instructions (Li-Cor Biosciences).