Peroxidase labeled horse anti mouse igg antibody

Protein concentration was determined using the bicinchoninic acid method. A total of 65 µl of each sample was mixed with 25 µl of NuPAGE LDS 4 × sample buffer (ThermoFisher Scientific, NP0007) and 10 µl of NuPAGE Sample Reducing Agent (ThermoFisher Scientific, NP0004) and heated at 70 °C for 10 min. Samples were run on 4–12% NuPAGE Bis–Tris gels (Invitrogen) and transferred to reinforced nitrocellulose membranes (Bio-Rad). After blocking with 5% fat-free milk in TBST buffer (20 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.6) for 60 min at room temperature, the membranes were incubated overnight with the following primary antibodies: rabbit anti-phospho-DRP1 (1:1000 dilution, Ser637, Cell Signaling, 4867), mouse anti-OPA1 (1:1000 dilution, BD Bioscience, 612,606), rabbit anti-FIS1 (1:500 dilution, FL-152, Santa Cruz, sc-98900), and mouse anti-VDAC1 (1:500 dilution, B-6, Santa Cruz, sc-390996). After washing, the membranes were incubated with peroxidase-labeled goat anti-rabbit IgG antibody (1:2000 dilution, Vector, PI-1000) or peroxidase-labeled horse anti-mouse IgG antibody (1:4000 dilution, Vector, PI-2000). Immunoreactive species were visualized using the SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, 34,580) and an LAS 3000 cooled CCD camera (Fujifilm, Japan).

Brain tissue from the parietal cortex in both hemispheres was dissected out and homogenized immediately on ice with a Dounce tissue homogenizer (Sigma, D8938) in tissue lysis buffer [15 mM Tris-HCl, pH 7.6, 320 mM sucrose, 1 mM dithiothreitol, 1 mM MgCl₂, 3 mM EDTA-K, and 0.5% protease inhibitor cocktail (Sigma, P8340)]. The homogenate was centrifuged at 4°C and 9,200× g for 15 min, and the protein concentration of the supernatant was measured using the bicinchoninic acid method. Individual samples of 20 μg protein were loaded and run on 4%–12% NuPAGE Bis-Tris gels (Invitrogen, Cat# NP0336BOX) then transferred to reinforced nitrocellulose membranes (Bio-Rad, Cat# 162-0112). Membranes were incubated with mouse anti-fodrin (1:1,000 dilution, Enzo Life Sciences, Cat# BML-FG6090-0500) and rabbit anti-actin (1:200 dilution, Sigma, Cat# A2066) overnight at 4°C. After washing, the membranes were incubated with peroxidase-labeled goat anti-rabbit IgG antibody (1:2,000 dilution, Vector, Cat# PI-1000) or peroxidase-labeled horse anti-mouse IgG antibody (1:4,000 dilution, Vector, Cat# PI-2000). Immunoreactive species were visualized using the Super Signal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, Cat# 34580) and a LAS 3000 cooled CCD camera (Fujifilm, Tokyo, Japan).