Rabbit anti bdnf antibody

Rats were anesthetized deeply with ether and perfused transcardially with 50 mL of saline, followed by 500 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The L3, L4, and L5 DRG were dissected and post-fixed in the same fixative for 30 minutes, incubated in phosphate-buffered 20% sucrose solution overnight. These tissues were embedded in OCT compound (Tissue-tek^®; Sakura Finetek, Tokyo, Japan) and 8 μm thick frozen sections were cut. For visualization of BDNF in the DRG, the avidin-biotin-horseradish peroxidase complex method was used. These sections were incubated overnight with rabbit anti-BDNF antibody (1:4000, #sc-546; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with biotinylated goat anti-rabbit IgG (immunoglobulin G) (1:200) and avidin-biotin-horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Immunoreaction products were visualized with diaminobenzidine and nickel ammonium sulfate. DRG neurons were categorized as small (<20 μm diameter), medium (20–40 μm diameter), and large (>40 μm diameter), and the proportion of BDNF-immunoreactive (IR) neurons was analyzed in each group. The cell counting was performed by a blinded observer. Three representative sections that contained over 70 neurons, total at least 210 each neuron, with distinct nuclei were randomly selected in each of the DRG.

ProBDNF digestion samples were run on 15% SDS-PAGE gels and electro-transferred onto polyvinylidene difluoride membrane (Immobilon-P, Millipore), using semi-dry method. Membranes were blocked for 2 hours at room tempertaure with 10% (w/v) dried non-fat milk powder in Tris-buffered saline with 0.1% Tween 20 (TBS-T). After blocking, the membranes were incubated at 4°C overnight with rabbit anti-BDNF antibody (Santa Cruz) diluted 1:500 in 5% (w/v) non-fat milk in TBS-T. Membranes were then incubated for 2 hours at room temperature with horseradish-peroxidase-labelled secondary antibody (Goat Anti-Rabbit IgG Antibody; Vector Laboratories) diluted 1:10,000 in 5% dried non-fat milk powder in TBS-T. After washing, the peroxidase activity was visualized on photographic film with ECL Prime Western Blotting Detection Reagent (Cytiva). The developed film was later digitized on ChemiDoc MP Imaging System (Bio-Rad) and analyzed using Image Lab software (Bio-Rad).

Western blotting for TrkB and BDNF protein was performed. The hippocampal tissues were homogenized on ice and lysed in a lysis buffer containing 50 mM HEPES (pH, 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, 1.5 mM MgCl₂·6H₂O, 1 mM sodium orthovanadate, and 100 mM sodium fluoride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Protein samples (30 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane. The membranes were incubated with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-TrkB antibody (#SC-8316, 1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-BDNF antibody (#SC-546, 1:1,000; Santa Cruz Biotechnology), and mouse anti-β-actin (#SC-47778, 1:1,000, Santa Cruz Biotechnology). Subsequently, membranes were incubated for 1 hr with appropriate secondary antibodies (1:2,000; Vector Laboratories, Burlingame, CA, USA), and band detection was performed using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) (Ko et al., 2018 (link)).