Monoclonal anti myosin myh7

SM samples were lysed in RIPA buffer using Precellys (Bertin Instruments, Bretonneux, France). Fifty micrograms of protein were separated by SDS-PAGE, transferred to a PVDF membrane, and incubated with the following anti-mouse antibodies: monoclonal anti-myosin (MYH7) (#M8421, 1:500; Sigma–Aldrich, St. Louis, MO), pAKT (Ser473) (#4051,1:1,000), AKT (#9272, 1:1,000), p4eBP1 (#2855, 1:1,000), and 4eBP1 (#9644, 1:1,000; all from Cell Signaling, Danvers, MA). GAPDH (#2118, 1:1,000; Cell Signaling, Danvers, MA) and α-tubulin (NB100-690, 1:1,000; Novus, Centennial, CO) antibodies were used as loading controls. HRP-conjugated anti-rabbit (#31460, 1:2,500; Thermo Fisher Scientific, Waltham, MA) and anti-mouse (P0260, 1:1,000; Dako, Glostrup, Denmark) were visualized using the Clarity™ Western ECL Substrate Kit on a ChemiDoc™ MP imaging system (both Bio-Rad Laboratories, Hercules, CA). pAkt/AKT and p4eBP1/4eBP1 ratios were estimated by densitometry (ImageJ® Software, Version 1.53r). MYH7 expression was normalized to the expression of GAPDH.

SM were carefully dissected, mounted on Tissue-Tek® O.C.T™ (Sakura Finetek, Hatfield, PA), and snap frozen in liquid nitrogen-cooled 2-methylbutane for 10–20 s. Samples were stored at −80 °C prior to cryosectioning. SM cryosections were washed with PBS and blocked with 0.05 % TBST (0.05 % Tween-20 in TBS) containing 10 % anti-goat serum. Slides were incubated with monoclonal anti-myosin (MYH7) (#M8421, 1:300; Sigma–Aldrich, St. Louis, MO) or anti-laminin antibodies (#PA1-16730, 1:500, Thermo Scientific, Waltham, MA) in blocking solution overnight at 4 °C. After washing with TBS, sections were incubated with secondary goat anti-rabbit Alexa Fluor-488 (#A-11008, 1:250) and anti-rabbit Alexa Fluor-594 (#A-11012, 1:250, both Thermo Fisher Scientific, Waltham, MA) antibodies in TBST plus anti-goat serum for 1 h at RT, followed by a 10-min incubation with DAPI. Slides were mounted with Dako Fluorescence Mounting Medium (Agilent Technologies, Santa Clara, CA) and visualized using an Olympus BX63 microscope equipped with an Olympus DP73 camera (Olympus, Shinjuku, Japan). The cross-sectional area (CSA) and Feret diameter of myofibers were determined using Fiji software (ImageJ® Version 1.52d; plugin “Muscle morphometry”). The areas of immunofluorescently stained fibers were quantified using ImageJ software (Version 1.53r).