For immuno-EM, cells were fixed with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4), and embedded in 12% gelatin. Blocks were infiltrated with 2.3 M sucrose for cryoprotection, mounted on specimen holders and frozen in liquid nitrogen. Cryosections were cut with a Leica EM UC6/FC6 Microtome (Leica Microsystems, Vienna, Austria). Thawed cryosections were labeled with anti-E 4G2 antibody, rabbit polyclonal antibody against ER resident protein calreticulin (Abcam, Cambridge, MA, USA) and protein-A gold (10 nm and 15 nm) obtained from Utrecht University (Utrecht, The Netherlands) and used as described before [45] . Double labeling was performed sequentially, using for each antibody a different size of protein A gold. Unspecific binding of the second protein A to the first antibody was blocked by incubation with 1% glutaraldehyde as described before [46] . The grids were viewed with Philip CM10 electron microscope at 80 kV and images were taken with KeenView camera (Soft Imaging System, Lakewood, CO, USA) using iTEM 5.0 software (Soft Imaging System GmbH).