Rabbit anti trkb

Animals were sacrificed after deeply anesthetized with isoflurane, total proteins from the lumbar enlargements of spinal cords were extracted by ice-cold Radio Immunoprecipitation Assay (RIPA) lysis. Proteins were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, United States). After blocking with 5% nonfat milk, the samples were incubated with rabbit anti-β-actin (1:3000, Abcam, United States), rabbit anti-GAPDH (1:3000, Merck Millipore, United States), mouse anti-VGluT2 (1:2000, Abcam, United States), goat anti-Iba-1 (1:2000, Wako, Japan), rabbit anti-GFAP (1:2000, Abcam, United States), rabbit anti-BDNF(1:2000, Abcam, United States), or rabbit anti-TrkB (1:2000, Abcam, United States) at 4 °C overnight. The next day, the corresponding rabbit, goat, or mouse Horseradish peroxidase (HRP)-conjugated secondary IgG (1:20000, Jackson ImmunoResearch, United States) was incubated for 1 h at room temperature. The gray value of the protein on the membrane of the corresponding molecular size was detected using western blotting detection kit (WesternBright Sirius, Advansta, United States) and ChemiDoc XRS imaging system (Bio-Rad). Image Lab 3.0 system (Bio-Rad) was utilized to perform standardized analysis on the test results.

The rats in each group (n = 3) were sacrificed at seven and 14 days after MCAO. Tissues in the ipsilateral hemisphere (excluding the infarct area) were selected for Western blotting. Under deep anesthesia, the brain tissue was rapidly removed and dissected, and protein was homogenized in a cell lysis buffer (Fermentas, Burlington, ON, Canada) containing a complete protease inhibitor cocktail (Thermo, Rockford, AL, USA). Next, 12% sodium dodecyl sulphate-polyacrylamide gels were used to separate equal amounts of protein from each sample which were electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore), The membranes were incubated with rabbit anti-BDNF (1:2000; Abcam, 15 kDa), rabbit anti-TrkB (92 kDa), phosphorylated-TrkB (91 kDa), AKT(56 kDa), phosphorylated-AKT (56 kDa, phospho S473), CREB (37 kDa), phosphorylated-CREB (37 kDa, phospho S133) (1:1000; Abcam), and mouse anti-α-tubulin (1:1000; Cell Signaling Technologies, 50 kDa) overnight at 4 °C after blocking with 5% skimmed milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at 25 °C. The membranes were then washed in TBST and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (1:10,000; Abcam). Immunoreactivity was visualized by using the enhanced chemiluminescence method.

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.