Lsm510 meta laser scanning confocal microscope system

EDL muscles were dissected from sacrificed animals, fixed in 2% paraformaldehyde in phosphate buffered saline (PBS) for 20 min at room temperature and stored at 4 °C overnight. Small bundles of fixed EDL fibers were washed three times in PBS containing 1% bovine serum albumin (PBS/BSA) and incubated in blocking solution (PBS/BSA with 10% goat serum and 0.5% Triton X-100) for 1 h at RT, followed by an overnight incubation at 4 °C with one of the following primary antibodies: a) mouse monoclonal anti-RYR1 (34C, dilution 1:30; Developmental Studies Hybridoma Bank University of Iowa, Iowa City, Iowa), b) rabbit polyclonal anti-translocase of outer mitochondrial membrane 20 homolog (TOM20, dilution 1:100; Santa Cruz Dallas, TX). Muscle bundles where then washed three times in PBS/BSA and then incubated for 1 hr at RT with the appropriate secondary antibodies: a) Cy5-labeled goat anti-mouse IgG (dilution 1:50); or b) Cy3-labeled goat anti-rabbit (dilution 1:200). All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (Lexington, KY). Confocal images were acquired using a Zeiss LSM510 META laser-scanning confocal microscope system (Zeiss, Jena, Germany) equipped with a Zeiss Axiovert 200 inverted microscope and a Plan Neofluar oil-immersion objective (100X/1.3 NA).