Anti sarcomeric α actinin

Muscle strips were removed from skeletons and rinsed in PBS. For fluorescent staining, tissues were fixed in 4% paraformaldehyde (Electron Microscopy Services) for 30 min and permeabilized with 0.25% Triton X-100 (Sigma-Aldrich) for 10 min. After blocking in Image-iT® FX (Molecular Probes) overnight at 4 °C, muscle strips were incubated with MF-20 (1:400, Developmental Studies Hybridoma Bank, The University of Iowa) and anti-sarcomeric α-actinin (1:400, Abcam) primary antibodies overnight at 4 °C, rinsed 3x with PBS and incubated with Alexa Fluor® 488 goat anti-mouse and Alexa Fluor® 568 goat anti-rabbit (both 1:400, ThermoFisher) overnight at 4 °C in the dark. Muscle strips were rinsed 3x with PBS, incubated with 4′,6-diamindino-2-phenylindole (DAPI, 1:5,000 in sterile DI water, Sigma-Aldrich) for 10 min, rinsed and imaged with a confocal microscope (LSM 710, Zeiss). For histology, frozen muscle strips were embedded in OCT compound (TissueTek), cut into 14 μm sections using a cryostat (CM3050, Leica), mounted on glass slides, stained with a Masson’s Trichrome kit (Polysciences, Inc.) and imaged using a digital pathology slide scanner (C9600, NanoZoomer).