2.9 Transmission Electron Microscopy (TEM)

EHT samples were kept on the posts with braces and fixed overnight in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer at room temperature, then stored for 1–3 days at 4 °C. Subsequently, fixed tissues were washed in buffer, then treated with 2% buffered osmium tetroxide for 1 hr on ice. Tissues were then washed in water at room temperature and dehydrated through a graded series of ethanol on ice, followed by two changes of propylene oxide at room temperature. Each tissue was infiltrated in a 1:1 mixture of propylene oxide and Epon Araldite epoxy resin overnight at room temperature. The next day the tissues were infiltrated with two changes of Epon Araldite, placed in flat embedding molds and polymerized overnight at 60 °C. Thin sections (80 nm) were taken and the sections were imaged using a JEOL 1230 TEM.