Horseradish peroxidase linked anti rabbit

The cortex and the hippocampus were gently homogenized in cold RIPA buffer (Radioimmunoprecipitation assay buffer; 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) on ice, centrifuged at 175,000×g for 1 h at 4 °C and the soluble supernatant was collected. The protein concentration was determined with Pierce BCA assay kit (ThermoFisher, USA). Equal amounts of total protein (50 μg per lane) were separated on 4~12% Bis-Tris-polyacrylamide electrophoresis gel and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies overnight at 4 °C: anti-NeuN (1:1000, Millipore, USA, CAT: MAB377), anti-Aβ (6E10; 1:1000, BioLegend, USA, CAT: 803002), anti-GFAP (1:1000, Millipore, USA, CAT: MAB360), anti-gp91 (1:1000, BD, USA, CAT: 611414), anti-iNOS (1:1000, Santa Cruz, USA, CAT: sc-7271), and anti-β-actin (1:1000, Cell Signaling Technology, USA, CAT: 8457). The membrane was then incubated with horseradish peroxidase-linked anti-rabbit (1:2000, Cell Signaling Technology, USA, CAT: 7074) or anti-mouse IgG (1:2000, Cell Signaling Technology, USA, CAT: 7076) for 1 h. The blots were detected with enhanced chemiluminescence (ECL) reagent. Images were analyzed by Image J software.