Electron micrograph of PC12 cells transfected with AP-3 siRNA. PC12 cells were plated onto aclar film discs (Pella) coated with poly-L-lysine, transfected twice with 50 nM AP-3delta1 siRNA and fixed with 2.5% glutaraldehyde in calcium-/magnesium-free PBS. The discs were washed three times with 0.1 M sodium cacodylate buffer, pH 7.2, and postfixed for 30 min on ice with 1% osmium tetroxide in cacodylate buffer containing 1.6% potassium ferricyanide. The discs were then washed three times with cacodylate buffer, three times with water, incubated with 0.5% uranyl acetate for 30 min (in the dark), and washed again with water. The samples were dehydrated in a graded series of ethanols while progressively lowering the temperature from 4°C to −40°C then embedded in epon resin. After peeling off the aclar, 60-nm sections were cut and viewed in an electron microscope (FEI Tecnai 12; Phillips) at 120 kV, images were captured with a digital camera at 6,800 magnification. Image is similar to Fig 6C right panel (CIL# 13764) of J Cell Biol. 2010. 191: 1173-1187.