Bdnf n 20

Retinas were excised from the eye cup immediately after death, and tissue was snap-frozen on dry ice before lysing using a Lysis-M reagent containing cOmplete Mini Protease Inhibitor (Roche) and phosphatase inhibitors (Thermo Fisher Scientific). Following 20-min homogenization, tissue was centrifuged at 13,000 rpm for 10 min to isolate the soluble cell extract. Protein concentration was determined using a bicinchoninic acid protein assay (Thermo Fisher Scientific), and equal quantities of protein were loaded onto 10% or 4 to 12% bis-tris gels (NuPAGE Novex, Thermo Fisher Scientific). Membranes were blocked in 5% dried skimmed milk in PBS with 0.2% Tween 20 (Sigma-Aldrich) for 60 min and then incubated overnight at 4°C in primary antibody (BDNF N-20, rabbit, 1:200, sc-546, Santa Cruz Biotechnology; TrkB, rabbit, 1:500, ab33655, Abcam; β-actin, rabbit, 1:1000, 4967, Cell Signaling Technology). Primary antibodies were visualized with horseradish peroxidase–conjugated anti-rabbit secondary antibody (1:8000; PI-1000, Vector Laboratories) and signal detection using ECL Prime (GE Healthcare) and an Alliance Western blot imaging system (UVItec Ltd.).