Propylene oxide

Following confocal scanning, each sample on the Aclar film was post-fixed with OsO4 solution (1% OsO4, 0.5% Potassium dichromate, 0.5% Potassium hexacynoferrate, in 0.1 M Cacodylate buffer) for 1 h at 25 °C. Samples were then washed 2 × 5 min in 0.1 M Cacodylate buffer followed by 4 × 3 min washes in DDW, stained with 2% Uranyl acetate in DDW, and covered with aluminum foil for 1 h. Thereafter, the samples were washed 4 × 5 min with DDW and dehydrated in a graded series of ethanol (30, 50, 70, 90, 95, 3 × 100%) 10 min each on ice. Samples were then incubated with propylene oxide (#00236, Polysciences Inc.) 3 × 5 min at room temperature, and transferred to 50% followed by 75% Epon (#08791-500, Polyscience Inc.) in propylene oxide, each overnight. Samples were then left in a hood for 4 h to allow the propylene oxide to evaporate, and then infiltrated with 100% Epon overnight and fresh Epon 3X2h. All the above steps were done without agitation. Finally, samples were embedded in a flat mold and polymerized at 60 °C for 48 h.