Anti trkc

Following anesthesia, the brains were removed from the skull and these were dissected into the right or left hemispheres. For protein extraction, they were placed in 10 volumes of cold homogenization buffer (120 mM NaCl, 50 mM Tris, pH 7.4) with protease inhibitors (Complete Mini, Gibco, Grand Island, NY, USA) being freshly added. Tissue then was homogenized by sonicator. Concentrations of protein were checked by the Bradford method (BioRad, Richmond, CA, USA). By adding the sampling buffer, equal amount of protein, 20 μg, was loaded and separated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used with 10% polyacrylamide and 0.05% bis-acrylamide [11 (link)]. By separating proteins on the gels, they were transferred into nitrocellulose membrane. They were probed with anti-NT-3 (1 : 300, Santa Cruz, CA, USA) and anti-trkC (1 : 300, Santa Cruz, CA, USA) as primary antibody. For secondary antibody, Peroxidase anti-rabbit IgG (Vector, PI-1000, 1 : 3000 dilution) was used. As an internal control, anti-β tubulin (1 : 300, Santa Cruz, CA, USA) was checked on the sample membrane. We detected signals with enhanced chemiluminescence (Supersignal, Pierce, Rockford, IN, USA), using autoradiogram by exposing 10 to 30 min [6 (link)–8 (link)].

At day 16, rats were anesthetized first and then sacrificed. For transcardiac perfusion, heparinized saline and then 4% paraformaldehyde in phosphate-buffered saline (PBS) were perfused. Sections were cut at a thickness of 30 μm using a sliding microtome. For blocking, 10% normal goat serum (NGS), 1% bovine serum albumin (BSA), 0.2% Triton X-100, and 1% H₂O₂ were used in PBS. Following washing with PBS (×3), anti-NT-3 (1 : 300, Santa Cruz, CA, USA) and anti-trkC (1 : 300, Santa Cruz, CA, USA) antibodies were used in 10% NGS and 1% BSA for overnight at cold room with temperature of 4°C. We used DAB kit (Dako, Carpinteria, CA) for immunoperoxidase labeling. They were evaluated and captured using an BX51 microscope (Olympus, Japan) [6 (link)–8 (link)].