Mouse anti synapsin 1

After DIV 12, hippocampal primary neurons were fixed for 30 min with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), washed with DPBS (Invitrogen) three times and then blocked with 5% normal donkey serum in 0.1% TBS-Triton (TBS-TX) buffer for 2 h at room temperature. Primary antibodies were diluted in blocking solution at 4°C using the following dilutions: 1:1000 rabbit anti-MAP2 (Millipore), 1:1000 mouse anti-MAP2 (Sigma), 1:1000 mouse anti-Synapsin-1 (Abcam), 1:500 rabbit anti-MeCP2 (Cell Signaling Technology) and 1:500 mouse anti-MeCP2 (Sigma). After incubation in primary antibodies overnight, coverslips were incubated in the appropriate secondary antibodies diluted in blocking solution for 2 h at room temperature.
The brain tissues were fixed 2 weeks after stereotaxic injection by vascular perfusion through the left ventricle of the heart with sequential delivery of 50 ml of saline and 60 ml of 4% paraformaldehyde in 0.1 M PB. Coronal brain sections (40 μm) were prepared and processed for immunostaining using the anti-DCX (Santa Cruz; 1:300) antibodies.

Glioma cells were plated on poly-d-lysine and laminin-coated coverslips (Neuvitro) at a density of 10,000 cells per well in 24-well plates. Approximately 24 h later, 40,000 embryonic mouse hippocampal neurons (Gibco) were seeded on top of the glioma cells and maintained with serum-free Neurobasal medium supplemented with B27, gentamicin and GlutaMAX (Gibco). After 2 weeks of co-culture, cells were fixed with 4% paraformaldehyde (PFA) for 30 min at 4 °C and incubated in blocking solution (5% normal donkey and goat serum, 0.25% Triton X-100 in PBS) at room temperature for 1 h. Next, they were treated with primary antibodies diluted in the blocking solutions overnight at 4 °C. The following antibodies were used: rabbit anti-homer-1 (1:250, Pierce), mouse anti-synapsin-1 (1:200) and chicken anti-MAP2 (1:500, Abcam). The coverslips were then rinsed three times in PBS and incubated in secondary antibody solution (Alexa 488 goat anti-chicken IgG; Alexa 568 goat anti-mouse IgG, and Alexa 647 goat anti-rabbit IgG, all used at 1:500 (Invitrogen) in antibody diluent solution for 1 h at room temperature. The coverslips were rinsed three times in PBS and then mounted with VECTA antifade mounting medium with DAPI (Vector Laboratories).