For FM5-95 labeling, we used procedures similar to those previously published (Branco et al., 2008). First, we replaced the chamber medium with pre-warmed HBS for >30 min. We then perfused the postsynaptic compartment and perfusion channel simultaneously using 10 μM of FM5-95 in a solution containing 90 mM KCl, CNQX (20 μM), and APV (50 μM) for 1 min at room temperature. The postsynaptic compartment and perfusion channel were then rinsed in HBS containing FM5-95 for an additional minute. The postsynaptic compartment and perfusion channel were then washed with HBS containing 0.5 mM Ca2+ and 10 mM Mg2+ to reduce dye loss from spontaneous vesicle release and Advasep-7 (1 mM, Biotium) to facilitate FM dye removal from membranes.
