Transmission Electron Microscopy Sample Preparation

Cells were prepared for TEM as described in [35 (link),62 (link),63 (link)]. Briefly, cells were fixed with 2% glutaraldehyde EM (GA; Electron Microscopy Science) in 50 mM phosphate buffer pH 7.2, 150 mM NaCl (PBS) for 2 h at 4°C, post-fixed with 1.2% potassium permanganate overnight at 4°C. Cells were then embedded in 2% low-melting-point agarose, dehydrated through an ethanol series, and passed through QY-2 (methyl glycidyl ether; Nisshin EM, Tokyo, Japan). Next, cells were embedded in Quetol 812 mixture (Nisshin EM Tokyo, Japan). Ultrathin sections were stained in 4% uranyl acetate and 0.4% lead citrate, and viewed with a TEM JEM-1400 (JEOL, Tokyo, Japan) at 100 kV.