Orius 832.10w ccd camera

For correlative light and platinum replica electron microscopy, cells were allowed to spread on IgG-coated glass pre-coverslips coated with a thin layer of gold through a locator grid (400 mesh, Ted Pella, Inc., Redding, CA). The gold layer provided the coverslips with a finder grating recognizable by both light and electron microscopy^{80 (link)}. The cells were extracted for 3 min with extraction solution containing 1% Triton X-100, 2% PEG (MW 35,000), 4 µM unlabeled phalloidin in M buffer (50 mM imidazole, pH 6.8, 50 mM KCl, 0.5 mM MgCl₂, 1 mM EGTA, and 0.1 mM EDTA). Samples were triple washed in M buffer with 4 µM unlabeled phalloidin and fixed in 2% glutaraldehyde in PBS buffer for 20 min. Then samples were triple washed in PBS and quenched with 5 mg/mL NaBH₄ in PBS for 10 min and again triple washed in PBS. Next, cells were stained with Alexa Fluor 488-phalloidin and imaged by spinning disk confocal microscopy. Samples for correlative platinum replica electron microscopy were processed as described previously^{81 (link),82 (link)}. Samples were analyzed using JEM 1011 transmission electron microscope (JEOL USA, Peabody, MA) operated at 100 kV. Images were captured by ORIUS 832.10 W CCD camera (Gatan, Warrendale, PA) and presented in inverted contrast.