Rabbit anti syp

Specified brain regions in the ipsilesional brain hemisphere, including the peri-infarct area and hippocampus, were freshly dissected in ice-cold PBS and lysed in RIPA lysis buffer. The homogenates were centrifuged (14000×g, 10 min, 4°C), and proteins were collected from the supernatant. Protein samples from each group were loaded onto SDS-PAGE gels (4%-12%), separated electrophoretically and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated overnight at 4°C with one of the following primary antibodies: rabbit anti-NeuN (1:5000, Abcam), rabbit anti-SYP (1:20000, Abcam), rabbit anti-GAP43 (1:1000, Abcam), or rabbit anti-GAPDH (1:2000, Sangon Biotech, Shanghai, China). After washes with Tris-buffered saline containing 0.1% Tween 20 (TBST), membranes were incubated with secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature and then washed again. Protein bands were detected with an enhanced chemiluminescence (ECL) kit (CWBIO, Beijing, China). An investigator blinded to the experimental groups quantified the optical density after normalization to GAPDH by using Gel Analysis V 2.02 software 9 (Clinx Science Instruments, Shanghai, China).

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.