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Following fixation, cells were permeabilized with 0.1% Triton X-100 detergent (Bio-Rad), blocked with 8% bovine serum albumin (InterGen, Burlington, MA) in phosphate-buffered saline (PBS) followed by an incubation period with anti– α -vesicular glutamate transporter (VGLUT)1 (Synaptic Systems, Goettingen, Germany), anti-synapsin (Synaptic Systems), and anti–PSD-95 (Millipore, Billerica, MA) following the manufacturer’s protocol, at 4°C