L11600 21

Muscles and other tissues were frozen in nitrogen-cooled isopentane and liquid nitrogen for histological and immunoblot assays, respectively. Longitudinal and transverse 8 µm cryosections of mouse skeletal muscles were prepared, fixed and stained with antibodies against DHPRα 1 (1:100), RYR1 (1:200), α-actinin (1:1000), DNM2-R2680 (1:200), MTM1-R2827 (1:200), pan-isoform BIN1-C99D (1:50) and BIN1-R2444 (1:100) antibodies. Nuclei were detected by co-staining with Hoechst (Sigma-Aldrich) for 10 min. Samples were viewed using a TCS SP5 laser scanning confocal microscope (Leica). Air-dried transverse sections were fixed and stained with H&E, SDH, NADH-TR or Sirius Red/Fast Green staining and image acquisition performed with a slide scanner NanoZoomer 2 HT equipped with the fluorescence module L11600-21 (Hamamatsu Photonics) or a DMRXA2 microscope (Leica). Cross-sectional area (CSA) was analyzed in H&E sections from TA skeletal muscle using FIJI image analysis software. CSA (μm²) was calculated (>500 fibers per mouse) from 4 to 7 mice per group. The percentage of TA muscle fibers with centralized or internalized nuclei was counted in >500 fibers from 4 to 6 mice using the cell counter plug-in in ImageJ image analysis software.

Air-dried transverse cryosections (8 μm) were fixed and stained with Hematoxylin and Eosin (HE) or SDH, and image acquisition performed with a slide scanner NanoZoomer 2 HT equipped with brightfield and the fluorescence module L11600-21 (Hamamatsu Photonics). Minimum feret's diameter was analyzed in wheat germ agglutinin (WGA) sections from TA mouse skeletal muscle, using a plugin developed in ImageJ – ‘MyoMage’. Minimum feret's diameter was calculated in >500 fibers per mouse. The percentage of TA muscle fibers with centralized or internalized nuclei was counted in >500 fibers using the cell counter plugin in ImageJ (http://rsb.info.nih.gov/ij/) or FIJI analysis software. Qualitative SDH staining analysis was performed. Fiber to fiber variation in intensity in SDH staining is normal and is indicative of the oxidative state of the fiber. SDH staining is normally relatively homogeneous within each individual fiber. Accumulation within the center or the periphery of a fiber of SDH staining indicates an abnormal distribution.