Frozen brains were homogenized in 100 mg tissue/ml cold lysis buffer (20 mM Tris, 0.25 M sucrose, 2 mM EDTA, 10 mM EGTA, 1% Triton X-100) and one tablet of Complete Mini protease inhibitor cocktail tablets/10 ml (Roche Diagnostics, Indianapolis, IN, USA). Homogenates were centrifuged at 100,000 × g for 40 minutes, supernatant was collected, and protein levels determined using the BCA protein assay reagent kit (PIERCE, Milwaukee, WI, USA). The levels of TNFα, IL-1β, IL-6 and MCP-1 in brains or sera were measured with TNFα, IL-1β, IL-6 and mouse JE/MCP-1 commercial enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN, USA), as described previously [39].
