Anti stim1

EDL muscles were fixed and processed for immunofluorescence as previously described^{51 (link)}. Primary antibodies used: a) mouse monoclonal anti-RyR1/RyR3 (34 C antibody, 1:30, Developmental Studies Hybridoma Bank); b) rabbit polyclonal anti-STIM1 (1:100, Sigma Aldrich); c) mouse monoclonal anti-STIM1 (1:50, BD Biosciences); or d) rabbit polyclonal anti-Orai1, (1:20, Thermo Scientific). Secondary antibodies used: a) Cy5-labeled goat anti-mouse IgG (1:50); or b) Cy3-labeled goat anti-rabbit (1:200). Confocal images were acquired using a Zeiss LSM510 META system equipped with a Zeiss Axiovert 200 inverted microscope and a Plan Neofluar oil-immersion objective (100X/1.3 NA). Fluorescence image profiles were obtained from LSM 3.0 image analysis software by Zeiss. Co-localization was determined by Imaris 7.2.3 software (Bitplane) and quantified by an evaluation of the Pearson’s correlation coefficient values.

Extensor digitorum longus muscles were dissected from euthanized animals, fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at RT, and stored at 4°C overnight. Small bundles of fixed EDL fibers were washed three times in PBS containing 1% [PBS/bovine serum albumin (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 antibody, 1:30, Developmental Studies Hybridoma Bank University of Iowa, Iowa City, Iowa); (b) rabbit polyclonal anti-STIM1 (1:100, Sigma–Aldrich, Milan, Italy); and (c) rabbit polyclonal anti-ORAI1 (1:20, Thermo Fisher Scientific, Waltham, MA, United States). Bundles of EDL muscles were then incubated for 1 h at RT with the following secondary antibodies (Jackson ImmunoResearch Laboratories, Lexington, KY, United States): Cy5-labeled goat anti–mouse immunoglobulin G (IgG) (1:100); and Cy3-labeled goat anti–rabbit IgG (1:200) for double labeling. 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 (100 × /1.3 NA).

EDL muscles were fixed for 20 min at room temperature in a fixative mix containing 2% paraformaldehyde and 0.5% glutaraldehyde (in PBS buffer). Small bundles of fixed fibers were blocked for 1 hour in PBS containing 1% bovine serum albumin (BSA), 10% goat serum, and permeabilized with 0.5% TritonX-100. After incubation with primary antibodies, secondary antibodies conjugated with Nanogold® particles (either goat anti-mouse or goat anti-rabbit; Nanoprobes) were applied for 2 h at 4 °C (1:100). Samples were then post-fixed in 1% glutaraldehyde PBS buffer at room temperature and incubated with reagents to enhance the signal (Goldenhance™ EM Formulation, Nanoprobes) for 5 minutes. Fiber bundles were finally post-fixed, embedded, and sectioned with standard EM protocols. Primary antibodies used: mouse monoclonal anti-RyR1/RyR3 34 C antibody, (1:30, Developmental Studies Hybridoma Bank); rabbit polyclonal anti-STIM1, (1:100, Sigma Aldrich); rabbit polyclonal anti-Orai1, (1:20, Thermo Scientific).