After removal of culture medium, UM-SCC-22A cells were fixed in 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 orthogonally to the cell monolayer with a diamond knife. Thin sections were visualized in a JEOL 1010 TEM. In immunoelectron microscopy studies, after fixing with 4% paraformaldehyde (PFA), cells were permeabilized with 0.1% Triton X-100 for 10 min at room temperature (RT), washed, and blocked with 1% BSA in PBS for 20 min. Corresponding primary antibodies (1:50 dilution) were added to cells and incubated overnight at 4°C. After washing with PBS, Nanogold (1.4 nm) (Nanoprobes)–conjugated anti-mouse or anti-rat Fab fragments (1:200) were incubated with cells for 1 hour at RT. After post-fixation with 1% glutaraldehyde in PBS for 10 min at RT, LI Silver enhancement was performed for 5 min. After rinsing with H2O, specimens were embedded in Epon 812, and sections were cut orthogonally to the cell monolayer with a diamond knife. Thin sections were visualized in a JEOL 1010 TEM.

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