Photoconversion of FM1-43

The photoconversion procedure was modified from a prior study (Harata et al., 2001). After FM1-43 staining, cultures on glass coverslips containing vHipp axons were fixed with 2% glutaraldehyde in 100 mM PBS for 20 min, and washed with glycine (100 mM in PBS) for 1 h, and then washed in ammonium chloride (100 mM in distilled water) for 5 min. After brief rinsing in PBS, axons were incubated in DAB (1 mg/ml in PBS) for 20 min. Fluorescence excitation light was then continuously applied for 10-20 min in DAB solution. Cultures were then washed in ice-cold PBS and processed for EM.

After DAB photoconversion, microslices cultures were briefly rinsed with PBS and then postfixed with 1% osmium tetroxide containing 0.8% potassium ferricyanide for 30 min. The coverslips were en bloc stained with aqueous 2% uranyl acetate for 20 min, dehydrated through an ascending series of ethanols, and embedded in Durcupan resin for 48 h at 60°C.

Glass coverslips were removed by submerging in liquid nitrogen. Ultrathin sections (60-90 nm) were cut and then stained with 1% methanolic uranyl acetate and 0.3% aqueous lead citrate and observed with a JEOL 1200EX transmission electron microscope.