Immunofluorescence labeling of muscle tissue

All frozen skeletal muscles were placed in a Leica cryostat and transverse sections of 10 μm were performed. As previously described by Gill et al.¹⁵, sections were blocked for 45 min in 10% goat serum diluted in PBS 1X solution and incubated overnight at 4 °C in primary antibodies for the following Myosin heavy chain (MyHC) isoforms: MyHCSlow (BA-F8, 1:100 dilution; Developmental Studies Hybridoma Bank, Iowa City, IA), MyHC2A (SC-71, 1:100 dilution; Developmental Studies Hybridoma Bank), and Laminin (Sigma L9393, 1:200 dilution; Sigma-Aldrich, St. Louis, MO). Secondary antibodies were then incubated at a 1:2000 dilution, using Alexa Fluor Goat anti-Mouse IgG2b 647 (for MyHCSlow), Goat anti-Mouse IgG1 555 (for MyHC2A) and Goat anti-Rabbit 594 (for Laminin)¹⁵. CD 31 (PECAM-1, 1:100; Thermo Fisher Scientific, Inc., MA) antibody was incubated with Laminin antibody and Alexa Fluor Goat anti-Rat 488 and Goat anti-Rabbit 594 were applied at 1:2000 dilution.