Mouse anti synaptophysin

The following antibodies were used: rabbit anti-KA2 [42 (link), 48 (link)] (generous gift by S. Heinemann and M.Darstein); mouse anti-synaptophysin (Synaptic Systems, Germany), mouse anti-CtBP2 (Becton Dickinson, Germany), mouse anti-Calbindin (Swant, Switzerland), mouse anti-protein-kinase C (Sigma, Germany), Fluorescein labeled peanut agglutinin (Vectorlabs Burlingame, CA, 200μg/ml), Alexa 647 coupled anti-CACNA1F, (Bioss, USA), Alexa-coupled secondary antibodies (Molecular Probes, Invitrogen),.

Cells were fixed between day 80 and 121 in prewarmed 4% paraformaldehyde (PFA, Sigma Aldrich)/4% sucrose (Sigma Aldrich) in PBS (Merck Millipore) for 10 min at room temperature (RT). Cells were permeabilized for 15 min in PBS/0.1% Tween-20 (Carl-Roth GmbH + Co. KG), incubated in quenching solution (100 mM glycine in PBS) for 30 min and blocked in 5% normal donkey serum (Jackson ImmunoResearch Inc.) for 1 hour at RT. Cells were incubated with primary antibodies over night at 4 °C. The following primary antibodies were used: chicken anti-microtubule-associated protein 2 (MAP2) (1:2000; Merck Millipore, AB5543), mouse anti-pan-axonal neurofilaments (SMI-312; 1:1000; Covance Inc.); mouse anti-synaptophysin (1:200; Synaptic Systems). After washing the cells 3 x with PBS-T, primary antibodies were labelled with secondary antibodies Alexa-Fluor 405 or 647 (1:500; Jackson ImmunoResearch Inc.) for 1 hr at RT. Cells were washed 2 x in PBS-T and once in PBS and mounted on glass slides with Mowiol (ThermoFisher Scientific).

For protein expression analysis, primary cortical neurons transduced with purified rAAV vectors were detached by pipetting and cellular pellets were lysed in RIPA buffer: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, and 1% Triton X-100 and protease inhibitors (Sigma), 1 mM PMSF pH 7.4. Samples were resolved in SDS-PAGE gels with different percentages. Western blot analysis was performed as previously described.^{54 (link), 55 (link)}The following antibodies were used: mouse anti-alpha-tubulin 1 : 1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-beta-actin 1 : 1000 (Sigma); anti-p53 1 : 500 (Santa Cruz Biotechnology); rabbit anti-DCX 1 : 500 (Santa Cruz); rabbit anti-PSD95 1 : 1000 (Cell Signalling, Beverly, MA, USA), rabbit anti-NMDAR1 1 : 100 (Chemicon), rabbit anti-NMDAR2A/B 1 : 500 (Chemicon), mouse anti-synaptophysin 1 : 500 (Synaptic Systems, Goettingen, Germany), rabbit anti-synaptotagmin 1 : 500 (Sigma) anti-GluR1 1 : 1000 (Upstate, Charlottesville, VA, USA). The quantitation of protein expression was determined after normalization to tubulin or actin by measuring the optical density of respective band blots using the Quantity One software (Bio-Rad, Hercules, CA, USA).