Ab66066

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS/PAGE) was performed using 10% polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes using a semi-dry transfer system (Bio-Rad, Hercules, CA, USA) and immunoblotted using rabbit anti-GPR37-N (1 μg/ml)², goat anti-A_2AR (AB_2571655; 1 μg/ml; Frontier Institute Co. Ltd), rabbit anti-synaptophysin (ab23754; 1 μg/ml; Abcam), mouse anti-PSD-95 (ab13552; 1 μg/ml; Abcam), mouse anti-SNAP-25 (ab66066; 1 μg/ml; Abcam) and rabbit anti-α-actinin (sc-15335; 0.5 μg/ml; Santa Cruz Biotechnology Inc., Dallas, TX, USA) antibodies. The primary antibodies were detected using a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (65–6120; 1/50,000; Pierce Biotechnology), HRP-conjugated goat anti-mouse IgG (31430; 1/20,000; Pierce Biotechnology), HRP-conjugated rabbit anti-goat IgG (61–1620; 1/20,000; Pierce Biotechnology). The immunoreactive bands were developed using a chemiluminescent detection kit (Thermo Fisher Scientific, Waltham, MA, USA) and detected with an Amersham Imager 600 (GE Healthcare Europe GmbH, Barcelona, Spain)^{46 (link)}.

The brains were washed with saline and sequentially fixed in 10% formaldehyde solution and then post-fixed in 15% and 25% sucrose solutions. Two-micrometer sections were cut by using a microtome instrument. Antigen retrieval was performed enzymatically for 20 min. To reduce non-specific antibody binding, the samples were blocked with the blocking agent [10% normal goat serum (Sigma, G9023) and 0.3% Triton X-100 in PBS] for 30 min at 37°C. After washing, tissue sections were incubated with mouse anti−SNAP25 monoclonal (Abcam, ab66066) as the primary antibody at 4°C. FITC−conjugated anti−mouse IgG (Sigma, F9137) were used as secondary antibody for 2 h at room temperature. The nuclei were counterstained with DAPI (Sigma, D9542). The slides were visualized using an AX70 Olympus fluorescence microscope (three in each group).