Rabbit anti synaptophysin monoclonal antibody

At 3, 7, 14, and 21 d after HI injury, rats were euthanized, and brains were harvested (n = 5 each group). Thalamic tissues were isolated from the right hemispheres on ice and stored at −80°C. Total protein was extracted from frozen thalamic tissue samples and denatured. Protein samples (50 µg) were separated by 4%–20% sodium dodecyl sulfate-polyacrylamide precast gel electrophoresis (Genscript, USA; 140 V for 1 h) and then transferred to polyacrylamide difluoride (PVDF) membranes (100 V for 60 min). Membranes were blocked in 5% nonfat milk for 2 h before overnight incubation at 4°C with primary antibody (mouse anti-PSD-95 monoclonal antibody, 1 : 1000, Abcam, USA; rabbit anti-synaptophysin monoclonal antibody, 1 : 5000, Abcam, USA; mouse anti-tubulin monoclonal antibody, 1 : 2000, Proteintech, USA). Then, the membranes were incubated with the appropriate secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody, 1 : 5000; Proteintech, USA) and developed by enhanced chemiluminescence reagents (Thermo Scientific Pierce; Thermo Fisher Scientific, Waltham, USA). The intensities for all the bands were analyzed by ImageJ software and were normalized to the tubulin signal.

After deparaffinization and heat-mediated antigen retrieval (EDTA-based pH 9.0 solution), as previously described, the tissue sections were blocked with goat serum for 30 min at 37°C and then incubated at 4°C overnight with primary antibody (mouse anti-myelin basic protein (MBP) monoclonal antibody, 1 : 400, Abcam, USA; rabbit anti-synaptophysin monoclonal antibody, 1 : 100, Abcam, USA; goat anti-Olig2 polyclonal antibody, 1 : 50, R&D, USA). Sections were washed three times with 0.1 M phosphate buffered saline (PBS) for 5 min before incubation with secondary antibodies (donkey anti-rabbit IgG, 1 : 500, Abcam, USA; donkey anti-mouse IgG, 1 : 500, Abcam, USA; donkey anti-goat IgG, 1 : 200, Abcam, USA) for 4 h at room temperature and 4′,6-diamidino-2-phenylindole (DAPI; 1 : 500, Sigma-Aldrich) sequentially for nuclear staining. Immunofluorescent images were visualized and photographed using a confocal laser-scanning microscope (C1; Nikon, Tokyo, Japan).