Fig. 3. Immuno-EM shows that whereas EGFR in nonstimulated cells localizes both within and outside rafts, TfR localizes outside rafts. To patch PLAP, HEp-2 cells were incubated with mouse anti-PLAP antibodies, followed by rabbit anti-mouse IgG and finally 5 nm protein A-gold. Each incubation was for 30 minutes on ice. Ultrathin cryosections of HEp-2 cells with patched PLAP (small gold particles) were labeled (large gold particles) to detect localization of EGFR (A-C) or TfR (D). Labeling for EGFR was found both outside (small arrowheads in A) and within (large arrowheads in B,C) PLAP patches. Labeling for TfR was mainly found outside PLAP patches. Bars, 100 nm.