Rabbit anti trkb antibody

Western blot for the expressions of Bax, Bcl-2, BDNF, TrkB, CREB, and p-CREB were performed, according to the previously described method (Kim et al., 2014 (link); Ko et al., 2009 (link)). 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 (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: mouse anti-β-actin antibody, mouse anti-Bcl-2 antibody, mouse anti-Bax antibody, rabbit anti-BDNF antibody, rabbit anti-TrkB antibody, rabbit anti-CREB antibody, rabbit anti-p-CREB antibody (1:1,000; Santa Cruz Biotechnology). Subsequently, membranes were incubated for 1 hr with attempt secondary antibodies (1:2,000; Vector Laboratories), and ban detection was performed using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology).

Western blotting for the determination of BDNF and TrkB was conducted, according to the previous method (Park et al., 2017 (link)). Hippocampal samples were homogenized on ice and lysed in a lysis buffer containing 50 mM Tris-HCl (pH, 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P40, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, and 100 mg/mL leupeptin. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Thirty micrograms of total protein were separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. The membrane was blocked with dehydrated milk, then incubated with mouse anti-β-actin antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BDNF antibody (1:1,000; Santa Cruz Biotechnology), and rabbit anti-TrkB antibody (1:1,000; Santa Cruz Biotechnology). After washing, horseradish peroxidase-conjugated, appropriate secondary antibodies were applied. Incubations were performed at room temperature. The bands were detected using the enhanced chemiluminescence detection system (Santa Cruz Biotechnology), and quantified using an Image-Pro Plus computer-assisted image analysis system (Media Cyberbetics Inc., Silver Spring, MD, USA).

According to a previously described method [13 (link),21 (link)], western blot was conducted. From the peri-infarct cortex and hippocampus, total protein was extracted using lysis buffer containing 150mM NaCl, 1mM phenylmethylsulfonyl fluoride, 1% Nonidet P40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 100-mg/mL leupeptin, 50mM Tris-HCl (pH, 7.5). Protein was quantified using a Bradford protein assay (Bio-Rad, Hercules, CA, USA). Protein, 30 µg, was separated on SDS polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Chicago, IL, USA). The membrane was blocked with skim milk, then membrane was incubated by mouse anti-β-actin antibody (1:1,000; Santa Cruz Biotechnology), rabbit anti-PSD-95 antibody (1:1,000; Abcam, Cambridge, UK), rabbit antisynaptophysin (1:1,000; Abcam), rabbit antiBDNF antibody (1:1,000; Alomone Labs, Jerusalem, Israel), and rabbit anti-TrkB antibody (Santa Cruz Biotechnology) at 4°C during overnight. After washing, species appropriate horseradish peroxidase-conjugated secondary antibodies were incubated for 1 hour. Band detection was performed using the enhanced chemiluminescence detection system (Santa Cruz Biotechnology).