RBL-2H3 cells were allowed to settle overnight onto 15-mm round, clean glass coverslips in the presence of anti-DNP IgE (1 μg/ml) to prime cell surface FcεRI. After washing to remove excess IgE, FcεRI was cross-linked by incubation with DNP-BSA (0.1–1 μg/ml), with rabbit polyclonal anti-IgE (1 μg/ml) or with gold conjugates of these reagents (prepared as in Seagrave et al. 1991). Plasma membrane sheets were prepared by a modification of a procedure described by Sanan and Anderson 1991. The coverslips were rapidly chilled by immersion in ice-cold Hepes buffer (25 mM Hepes, pH 7, 25 mM KCl, and 2.5 mM MgAcetate) and inverted onto nickel EM grids that had been coated with formvar and carbon and, on the day of the experiment, glow-discharged and floated on poly-l-lysine (0.8 mg/ml for 30 min, followed by 10 s dH2O rinse and air drying). Pressure was applied to the coverslip for 20 s by bearing down with a cork. The coverslips were lifted, leaving sections of the upper cell surface adherent to the poly-l-lysine–coated grid. Membranes were rinsed in 4°C Hepes buffer and fixed in 2% paraformaldehyde for 10 min. For experiments using anti–IgE-gold to label FcεRI in the resting state, cells were fixed with 0.5% paraformaldehyde for 5 min and then incubated with anti–IgE-gold before inversion onto EM grids. All membranes were labeled from the inside by inverting the grids onto droplets containing primary antibodies or biotin-phalloidin (5 units/ml) followed by gold-conjugated secondary reagents. Incubations were for 30 min. Intermediate washes in PBS were performed by inverting the grids onto droplets. Primary antibodies were diluted in PBS, 0.1% BSA at the following concentrations: Syk, 10 μg/ml; FcεRI β, 28 μg/ml; Lyn, 2 μg/ml. Gold-conjugated secondary reagents were diluted 1:20 from commercial stocks in PBS-BSA. The samples were post-fixed in 2% glutaraldehyde in PBS and held overnight in PBS. Next, samples were stained for 10 min with 1% OsO4 prepared in 0.1 M cacodylate buffer and washed 5 min with cacodylate buffer and twice for 5 min in dH2O. Samples were then processed for 10 min in 1% aqueous tannic acid, followed by two 5-min rinses with dH2O, 10 min with 1% aqueous uranyl acetate and two 1-min rinses with dH2O. Grids were air-dried and examined using an Hitachi 600 transmission electron microscope.