Rabbit anti bdnf antibody

After the tissues of the brain were harvested for western blot, PMC areas were lysed and extracted in a radioimmunoprecipitation (RIPA) buffer containing protease (Protease Inhibitor Cocktail Set I; Calbiochem, Darmstadt, Germany) and phosphatase inhibitors according to the manufacturer's instructions at 4°C. Then, the protein concentrations were determined by the BCA standard. After the preparation of PAGE gels, we loaded the samples, markers, and the loading control according to the comparison demand. Then, protein samples were separated by SDS-PAGE in 12% polyacrylamide gels and electroblotted onto a nitrocellulose membrane (Millipore, USA). After blocking in buffer for 2 h at room temperature, membranes were incubated with rabbit anti-BDNF antibody (1 : 1000) from Abcam, UK. Then, after being washed with PBST for 3 times, membranes were incubated at room temperature for 2 h with the corresponding horseradish peroxidase-conjugated secondary antibody diluted 1 : 1000 in blocking buffer. After washing, signals were detected by enhanced chemiluminescence (ECL Plus; Amersham Biosciences) on Kodak Biomax Light films. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were used as the loading control. The assays were performed thrice independently.

After the mice were sacrificed and perfused with 4% paraformaldehyde, the lumbar enlargements of the spinal cord were collected and dehydrated with 15 and 30% sucrose. The processed spinal cord specimens were then fixed with an OCT embedding agent and sliced using a Leica CM1860 cryostat. The sections were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS). After blocking with 5% donkey serum albumin for 1 h, the sections were incubated with the following primary antibodies: mouse anti-VGluT2 antibody (1:200, Abcam, United States), rabbit anti-NeuN (1:200, Abcam, United States), goat anti-Iba-1 (1:200, Wako, Japan), rabbit anti-GFAP (1:200, Abcam, United States), rabbit anti-SYP (1:200, Abcam, United States), rabbit anti-BDNF antibody (1:200, Abcam, United States), and rabbit anti-TrkB (1:200, Abcam, United States) overnight at 4°C. On the next day, the slides were resined in PBS, and then incubated with the corresponding goat, rabbit, or mouse Alexa Fluor 488 or 594-conjugated secondary IgG antibodies (1:200, Jackson ImmunoResearch, United States) in the dark. The slides were rinsed with PBS and mounted with DAPI Fluoromount-G (SouthernBiotech, United States). Images were obtained using a Leica Observer microscope. The images shown in the figures are representative results from three animals per group.