Immunogold staining of EGFR using electron microscopy.

A. Control and SPIN90 knockdown (KD) HeLa cells were cultured for 24 h and suspended in serum-free medium for an additional 1 h. Cells treated with 40 ng/ml EGF were incubated for 10 min to allow EGFR internalization, and fixed with 4% paraformaldehyde. Cells were prepared for electron microscopy, as described in Material and Methods. Sectioned samples were stained with mouse anti-EGFR antibody and labeled with 25 nm gold particles conjugated to anti-mouse antibody. B. Numbers of total endosomes per image (4.8 µm×6.0 µm) (a), numbers of endosomes with EGFR (b), numbers of EGFR localized at endosomes (c) and average EGFR numbers per endosome (d) were analyzed.