Anti bace1

The hippocampus was separated from the brain tissue and homogenized and lysed by adding a protein extraction solution (PRO-PREP, iNtRON, Sungnam, South Korea). After measuring the total protein concentration using Bradford reagent (Bio-Rad, Hercules, CA, USA), western blotting was performed as previously described (Han et al., 2019 (link)). Protein-specific primary antibodies were purchased from Novus Biologicals (anti-APP; Littleton, CO, USA), Santa Cruz Biotechnology (anti-GFAP and anti-β-actin; Dallas, TX, USA), Cell Signaling Technology (anti-iNOS, anti-COX-2, anti-p65, and anti-p50; Danvers, MA, USA), and Abcam [anti-BACE1, anti-ionized calcium-binding adapter molecule 1 (IBA-1), and anti-Foxp3; Cambridge, MA, USA]. Secondary antibodies were purchased from Santa Cruz Biotechnology (anti-mouse, anti-rabbit, and anti-goat).

Western blotting was performed as described in our previous study [19 (link)]. Total protein samples were extracted from brain tissues, and their protein concentrations were evaluated by the bicinchoninic acid (BCA) method (Bio-Rad, Hercules, CA, USA). Protein electrophoresed on 10% (w/v) SDS polyacrylamide gels were transferred onto polyvinylidene difluoride (PVDF) membranes, and blocked with TBST (TBS with 0.1% Tween-20) containing a 5% nonfat dry milk or 5% bovine serum albumin (BSA) for 1 h. After blocking, membranes were kept overnight at 4 °C in blocking solution containing different primary antibody, including anti-APP (1:6000, Abcam, Cambridge, MA, USA), anti-BACE-1 (1:10,000, Abcam, Cambridge, MA, USA), anti-MT1 (1:1000, IBL, Hamburg, Germany), anti-MT2 (1:6000, Abcam, Cambridge, MA, USA), and anti-β actin (1:50,000, CSL, USA) antibodies. The membranes were exposed to secondary antibodies after being washed with TBST at room temperature for 1 h. Immunoreactivity was detected using enhanced chemiluminescence (ECL) reagents. Quantitative results were performed using a FusionCapt Advance Camera and FusionCapt Advance Analyzer (version 16.07, Sursee, Switzerland).