Surface Labeling and Immunofluorescence of Neurons

GluA1 (1:4; Calbiochem, PC246) and GluA2 (1:100; Millipore, MAB397) surface labeling was performed as previously described^{20 (link),38 (link)}. Briefly, cultures were washed with PBS containing 0.5 mM CaCl₂ and 1 mM MgCl₂ (PBS^MC) with 4% sucrose. Neurons were preincubated at 37° C for 5 min with primary antibodies against GluA1 to allow labeling of surface AMPA receptors, washed with ice-cold PBS^MC, fixed with 4% PFA + 4% sucrose for 15 minutes, then blocked in a detergent-free blocking solution (PBS with 2% normal goat serum (Sigma, St. Louis, MO), and 0.02% sodium azide) for 1 hr, followed by secondary antibody incubation at room temp for 1 hour. To immunolabel for excitatory synaptic markers, cultures were then postfixed with –20°C methanol for 1 min to permeabilize the neurons. Cells were blocked in blocking solution for 1 hr, followed by incubation with primary antibodies for PSD95 (1:200; Pierce, 6G6-1C9) and VGluT1 (1:500; Millipore AB5905). Neurons were washed, then incubated with a secondary antibody at room temp for 1hr.