Rabbit anti bdnf

Western blotting was performed as described previously (Bellanger et al, 2011 (link)). Rabbit anti-BDNF, anti-NGF, anti-NT3, anti-p75^NTR, goat anti-sortilin, and mouse anti-PARP-1 antibodies were purchased from Santa Cruz Biotechnology. Mouse anti-TrkB, anti-TrkA, and anti-TrkC antibodies were purchased from R&D Systems. Anti-phospho TrkB (pY817) and anti-phospho-TrkC (Y820) were purchased from Abcam (Epitomics, Burlingame, CA, USA) and anti-phospho-TrkA (Y490) from Cell Signalling. Protein-loading control was performed with anti-βactin Ab (Sigma). Visualisation of immunocomplexes was accomplished using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Darmstadt, Germany). Western blots were scanned using a bioimaging system (Genesnap; Syngene Europe, Cambridge, UK).
Co-immunoprecipitations were performed in accordance with the manufacturer's instructions (Catch and Release kit, Millipore) with 500 μg of proteins and anti-p75^NTR for 1 h at room temperature. Finally immunoprecipitates were subjected to SDS–polyacrylamide gel electrophoresis, before analysis by western blotting (with anti-Trk, anti-sortilin and anti-p75^NTR).

Western blot analysis for BDNF, TrkB, and AMPK was done as previously explained method [14 ,22 (link)]. Lysis buffer was applied to lyse hippocampal tissues, and protein content was detected by a colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Sodium dodecyl sulfate-polyacrylamide gel was used to separated 30-μg protein, then reaction mixture was transferred to a nitrocellulose membrane, stopped reaction by applying dehydrated milk, and then incubated by primary antibodies. Mouse anti-β actin (1:3,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BDNF (1:1,000; Santa Cruz Biotechnology), rabbit anti-TrkB (1:1,000; Santa Cruz Biotechnology), and rabbit anti-AMPK (1:1,000; Santa Cruz Biotechnology) were selected as the primary antibodies. Horseradish peroxidaseconjugated secondary antibodies were used, and enhanced chemiluminescence detection system (Santa Cruz Biotechnology) measured the expression of bands.

Following decapitation, rat hippocampus was isolated rapidly and stored at −80 °C for Western blotting detection. The tissues were weighed, sonicated in RIPA lysis buffer supplemented with fresh protease and phosphatase inhibitors, and centrifuged at a speed of 12,000 rpm for 25 min. Then the protein concentration was determined by BCA assay. After denaturation and electrophoresis, the proteins were transferred onto a PVDF membrane in wet conditions. Following blocking in Tris-buffered saline Tween-20 solution with 5% (w/v) nonfat milk powder (1 h, RT), the protein membranes were incubated respectively in primary antibody: rabbit anti-BDNF (1:400, Santa Cruz Biotechnology, Inc., CA, USA), cAMP response element binding protein (CREB, 1:1000, Cell Signaling Tech), p-CREB (Ser133, 1:400, Cell Signaling Tech, MA, USA) and β-actin (1:8000, Sigma–Aldrich Inc., MO, USA) at 4 °C overnight. After washing, the membranes were incubated (1 h, RT) with anti-rabbit or anti-mouse IgG labeled with horseradish peroxidase (Jackson ImmunoResearch Inc., PA, USA) and then the membranes were washed and developed with Chemiluminescence reagents (Millipore Corp., MA, USA). The chemiluminescence signal was transformed into a digital image using FUJIFILM imaging system LAS-3000 (Fuji Photo Film Co., Ltd., Tokyo, Japan).