Horseradish peroxidase hrp conjugated anti mouse or anti rabbit secondary antibody

To prepare protein extraction, the frozen brains were homogenized in T-PER™ reagent (Tissue Protein Extraction Reagent, #78510, Thermo Fisher Scienti c/Invitrogen, Carlsbad, CA) supplemented with a cocktail of protease inhibitors (Complete™ Protease Inhibitor Cocktail, Roche/Sigma Chemical Co., St. Louis, MO), followed by centrifugation at 10000 × g for 10 minutes to harvest the supernatant. 50 µg total protein for each sample was loaded in 12% SDS gel. After electrophoresis, the protein was transferred to polyvinylidene di uoride (PVDF) membranes. After blocking for 1 hour with blocking buffer (5% of BSA in TBST), the membranes were probed with primary antibodies overnight at 4 °C. The following antibodies were used: IL-1β (1:500; #500-P80; Peprotech, New Jersey, USA) and GAPDH (1:50000; #6004-1-lg; Proteintech, Chicago, USA). After washing, the membranes were incubated with horseradish peroxidase (HRP) conjugated anti-mouse or anti-rabbit secondary antibody (Jackson ImmunoResearch, PA, USA) at room temperature for 1 hour. The blots were developed using chemiluminescence, captured by CCD chemiluminescence imaging system (ChemiDoc XRS Imaging System, Bio-Rad, Hercules, CA) and the intensity of each band was quanti ed using NIH Image J software.