Immunohistochemistry
myosin, Ti2pad
SECONDARY, -20
Muscle samples embedded in the OCT compound were cut into 5 μm sections in a cryostat at -20°C and transferred to the Super Frost Plus microscope slide (Fisher brand, Pittsburgh, PA, USA). Tissue sections were fixed in 4% paraformaldehyde solution for 5 min, permeabilized in 100% methanol for 10 min at– 20°C and 1% triton/phosphate-buffered saline (TPBS) for 10 min. Tissue sections were blocked for 30 min at room temperature in 10% goat serum in TPBS. To assess fiber type dependent CSA, the blocked tissue sections were incubated with primary antibodies against myosin heavy chain (MHC) I (BA.D5, 1:50, DSHB, Iowa City, IA, USA), MHC II (SC-71, 1:50, DSHB, Iowa City, IA, USA), and laminin (ab11575, 1:100, Abcam, Cambridge, MA, USA), and for analysis of the number of SC and Myostatin-positive SC (MSTN+/PAX7+), Pax7 (MAB1675, 1:100, R&D Systems, Minneapolis, MN, USA), Myostatin (AB3239, 1:100, Millipore, Etobicoke, ON, Canada), MHC II (ab91506, 1:1000, Abcam, Cambridge, MA, USA), and laminin (ab11575, 1:100, Abcam, Cambridge, MA, USA) were applied for overnight at 4°C. Appropriate secondary antibodies were applied; for CSA analysis, Alexa 647 goat anti-mouse IgG2b (A21242, 1:250, Invitrogen, Waltham, MA, USA), Alexa 488 goat anti-mouse IgG1 (A21121, 1:500, Invitrogen, Waltham, MA, USA), and Alexa 350 goat anti-rabbit IgG (H+L) (A11046, 1:500, Invitrogen, Waltham, MA, USA), and for SC and MSTN+/PAX7+, Alexa 594 (A11005, 1:500, Invitrogen, Waltham, MA, USA), Alexa 647 (A21244, 1:500, Invitrogen, Waltham, MA, USA), and Alexa 488 (A11008, 1:500, Invitrogen, Waltham, MA, USA). Nuclei were stained with 4,6-diamidino-2-phenylindole (1:2000, Sigma-Aldrich, Oakville, ON, Canada). Stained samples were imaged by a Nikon Eclipse Ti2pad microscope (Nikon Instruments, Melville, NY, USA) with a high-resolution Photometrics Moment 7 Megapixel CMOS camera (Teledyne Photometrics, Tucson, AZ, USA) with 20 times objective and analyzed using Nikon NIS element AR software (Nikon Instruments). All analyses were conducted on more than 100 fibers per fiber type per participant per time point [