The organization of endoplasmic reticulum export complexes

Cells were fixed in 2.5% glutaraldehyde (GA) in PBS for 1 h at room temperature (rt), scraped in GA, and pelleted at 15,000 g in a microcentrifuge for 10 rain. The tight pellet was washed in veronal-acetate buffer (pH 6.0) and stained in 1% buffered OsO4 for 1 h at rt. After washing in veronalacetate buffer, pellets were stained en bloc with 2% uranyl acetate in veronal-acetate buffer, dehydrated in alcohol and acetone, and embedded in Epon 812 (Electron Microscopy, Sciences, Fort Washington, PA) . For serial sectioning, the cell pellet was cut with a glass knife followed by trimming of the resulting block to obtain an ~40 ×300 ~xm pyramid. A ribbon of 25-35 consecutive sections of 65 nm in thickness was cut with a diamond knife on Reichert ultramicrotome 2E and transferred to a single 2 × 1.5 mm slot grid (Electron Microscopy Sciences) precoated with a Formvar/carbon film. Sections were counterstained with a saturated solution of uranyl acetate in methanol for 10 rain at rt and Reynold's lead citrate for 10 min. Budding structures on the ER and VTCs were followed in consecutive sections, and images were overlaid to reconstruct continuity between structures.