EDL muscles were dissected from sacrificed mice and fixed with paraformaldehyde 2% for 1-2 hrs at room temperature. Small bundles of muscles were processed for double immunostaining as previously described [23 (link)]. Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Secondary antibodies used (a) Cy5-labeled goat anti-mouse IgG and (b) Cy3-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Images were acquired using a Zeiss LSM510 META laser-scanning confocal microscope system (Zeiss, Jena, Germany) equipped with Zeiss Axiovert 200 inverted microscope and a Plan Neofluar oil-immersion objective (63X/1.3 NA). Negative controls for each immunostaining assay were performed by immunolabeling of samples with only secondary antibodies.
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