Nanogold 1.4 nm

After removal of culture medium, T cells were and rinsed with 1X PBS and fixed in freshly prepared 2% (w/v) glutaraldehyde in 0.1 M cacodylate buffer. After post-fixation in 2% (v/v) osmium tetroxide, specimens were embedded in Epon 812, and sections were cut orthogonal to the cell monolayer with a diamond knife. Thin sections were visualized in a JEOL 1010 transmission electron microscope (TEM). In immunoelectron microscopy studies, activated T cells were collected and rinsed with 1X PBS. They were fixed for 15 min with 4% paraformaldehyde, and cell permeabilization was achieved with 0.1% Saponin for 10 min, washed twice, and blocked with 1% BSA in PBS for 20 min. Primary antibodies (1:50 dilution) were added to cells and incubated overnight at 40C. After washing with 1% BSA PBS, Nanogold (1.4 nm) (Nanoprobes) conjugated donkey anti-mouse, or anti-rat Fab fragments (1:200) were incubated with cells for 1 h. After post-fixation with 1% glutaraldehyde in PBS for 10 min at RT, Li Silver enhancement was performed for 5 min. After rinsing with distilled H2O, enhanced with silver staining before specimens were embedded in Epon 812, and sections were cut orthogonal to the cell monolayer with a diamond knife. Thin sections were visualized in a JEOL 1010 TEM.