Immunolabeling.

Myotubes of the homozygous dysgenic (Ca_V1.1^mdg/mdg) cell line GLT were cultured as described in Powell et al. (37). At the onset of myoblast fusion (2 d after addition of differentiation medium), GLT cultures were transfected by using FuGene-HD according to manufacturer’s instructions (Promega). Cultures were fixed with paraformaldehyde 5 d after the transfection and double immunolabeled with rabbit anti-GFP (serum, 1:10,000; Molecular Probes) and mouse monoclonal anti-RyR (34-C; 1:1,000; Thermo Scientific) and fluorescently labeled with Alexa 488- and 594-conjugated secondary antibodies (Thermo Scientific), respectively. Samples were observed by using a 63×, 1.4-NA objective Axioimager microscope (Carl Zeiss Inc.), and 14-bit images were acquired with a cooled CCD camera (SPOT; Diagnostic Instruments) and Metaview image processing software. Images were arranged in Adobe Photoshop, and, where necessary, linear adjustments were performed to correct black level and contrast.