QD staining of surface GluN2B was performed as previously described (67, 68), with some modifications. Briefly, rabbit anti-NMDAR 2B (GluN2B) (1:10; AGC-003, Alomone Labs) antibodies were premixed with anti-rabbit QD 655 (1:15; Q11422MP, Life Technologies) in PBS for 30 min, and the solution was supplemented with casein (threefold concentrated, Vector Labs) during the last 10 min of incubation to prevent nonspecific binding. Neurons were then incubated with the diluted antibody-QD premix [1:50, in 148 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose, and 10 mM Hepes (pH 7.4)] for 3 min at room temperature.

Excitatory synapses were identified by transfecting neurons with PSD95-FingR-GFP, a disulfide-free intrabody that allows visualizing excitatory synapses in real time, in living neurons, with high fidelity and without affecting neuronal function (69). SPT experiments were performed using an inverted widefield microscope (Zeiss AxioObserver Z1 microscope) equipped with a 63× 1.4 NA oil immersion objective and a digital CMOS camera (ORCA-Flash4.0). The microscope was equipped with a large stage incubator, allowing to keep the cells at 37°C during the experiments. QD fluorescence was monitored over time by acquiring 1200 consecutive frames with an acquisition time of 50 ms using the ZEN Blue software 2012 (excitation filter BP 425/50 and emission filter BP 655/30, Chroma).

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