Immunogold electron microscopy was performed as previously described (39). Briefly, mice were anesthetized by isoflurane inhalation, and heparin was injected at 100 to 200 U per mouse. Hearts were excised and retrograde perfused with a solution containing 118 mM NaCl, 4.8 mM KCl, 25 mM Hepes, 1.25 mM K2HPO4, 1.25 mM MgSO4, and 11 mM glucose (pH 7.4). Mice were perfusion fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and hearts were dissected. For immunoelectron microscopy, dissected mice hearts were further fixed in freshly prepared 3% paraformaldehyde in 0.1 M phosphate buffer containing 0.1% glutaraldehyde and 4% sucrose (pH 7.4). Tissues were washed, dehydrated, embedded in Lowicryl K4M (Polysciences, Inc., Warrington, PA), and polymerized under ultraviolet light (360 nm) at −35°C. Ultrathin sections were cut and mounted on Formvar-carbon–coated nickel grids. After incubation with primary antibodies at 4°C overnight, gold-conjugated secondary antibodies [15-nm Protein A Gold, Cell Microscopy Center, University Medical Center Utrecht, 35584 CX Utrecht, The Netherlands; 18-nm Colloidal Gold-AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson ImmunoResearch Laboratories Inc., West Grove, PA] were applied and stained with uranyl acetate and lead citrate by standard methods. Stained grids were examined under Philips CM-12 electron microscope (FEI; Eindhoven, The Netherlands) and photographed with a Gatan (4k × 2.7k) digital camera (Gatan Inc., Pleasanton, CA).

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