Ultracut j microtome

Cells were seeded onto a 60-mm dish at 1×10⁶ cells/dish and treated with various reagents for 48 h. Then, the cells were fixed for 1 h at 4°C with 2.5% glutaraldehyde (TAAB Laboratories Equipment, Ltd.) in 0.1 M phosphate buffer (pH 7.3) (prepared with monobasic sodium phosphate and dibasic sodium phosphate; Wako Pure Chemical Industries), and fixed subsequently for 1 h at RT in 1% osmium tetroxide (Nisshin EM Co., Ltd.), dehydrated in graded ethanol (30-100%), and embedded in Quetol 812 epoxy resin (Nisshin EM Co., Ltd.) at 60°C for 2-4 days. ultrathin sections (60 nm) were obtained using an Ultracut J micro-tome (Reichert Jung), and the sections were stained with 4% lead nitrate (RT, 5 min) and saturated uranium acetate (RT, 10 min) and imaged using a transmission electron micro-scope JEM-1200EX II (JEOL, Ltd.) (magnification ranging from ×1,000 to ×10,000). All images were captured on films (Electron-microscopic film FG; Fujifilm).

TEM analyses were performed at the Hanaichi Ultrastructure Research Institute (Aichi, Japan). CAL27 cells (1×10⁶) were seeded into a 60-mm dish and pre-cultured for 24 h. Cells were treated with or without BTZ (5 nM) and/or RCS (5 µM) for 24 h, and then fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 16 h at 4°C. The samples were further fixed in 2% osmium tetroxide for 2 h at 4°C, dehydrated in graded ethanol (30, 50, 70, 90, 100, 100 and 100%) for 15 min each at 4°C, and embedded in Quetol 812 epoxy resin (Nisshin EM Co., Ltd.) for 48 h at 60°C. Ultrathin sections (80–90 nm) were cut using an Ultracut J microtome (Reichert Jung), stained with lead nitrate and uranium acetate, and imaged using an H-7600 transmission electron microscope (Hitachi High-Technologies Corporation) at 100 kV.