FM4-64 labeling

A fixable analog of FM4-64FX (Invitrogen) was diluted to 5 μg/ml working solution in high K⁺ solution (72mM NaCl, 50mM KCl, 1mM NaH₂PO₄, 26mM NaHCO₃, 1.8mM CaCl₂, 0.8mM MgSO₄, 11mM glucose and 20mM HEPES, pH-7.35). Day 12 neurons were incubated with FM4-64FX for 1 min at room temperature and washed three times using HBSS without magnesium or calcium. Neurons were then fixed with 4% PFA for 15 min and stained with phalloidin before mounting on glass slides for imaging.