Anti laminin α2

Transverse 7-μm-thick cryosections of quadriceps, heart, and diaphragm muscles were collected and stained with hematoxylin and eosin (H&E) and Masson’s trichrome stains (Sigma). For immunohistochemistry, cryosections were fixed with 4% paraformaldehyde/PBS or cold acetone. After blocking with 0.2% BSA/PBS, samples were incubated with primary antibodies at 37 °C for 1 h. Alexa Fluor 488 or 568 secondary antibody (1:1000; Thermo Fisher Scientific, Waltham, MO) with DAPI solution was used for detection. Primary antibodies used were as follows: anti-F4/80 (Bio-Rad), anti-embryonic myosin heavy chain (eMyHC, F1.652, Developmental Studies Hybridoma Bank, Iowa City, IA), anti-Pax7 (Developmental Studies Hybridoma Bank), anti-laminin α2 (Enzo Life Sciences, Farmingdale, NY), anti-Ki67 (Thermo Fisher Scientific), anti-PDGFRα (R&D Systems), and anti-periostin (Novus Biologicals). Anti-mouse IgG (H + L) secondary antibody (Alexa Fluor 488) was used to detect necrotic muscle fibers.

TA muscles were isolated from mice, washed in saline, and immediately frozen in isopentane chilled in liquid nitrogen. The frozen muscles were sliced into 10 µm thick cryosections. The cryosections were placed on slides and fixed in chilled acetone (−20 °C) for 10 min, followed by washing in PBS twice. The fixed cryosections were blocked with blocking buffer (5% goat serum and 1% BSA in PBS) for 15 min at room temperature, and treated with a diluted primary antibody [1/200 anti-Laminin-α2 (ENZO, Farmingdale, NY, USA)] in blocking buffer at 4 °C overnight, followed by incubation with 1:500 diluted Goat Anti-Rat Alexa 488 (Thermo Fisher Scientific) as a secondary antibody at room temperature for 2 h. After washing and staining with DAPI, the processed cryosections were examined by a fluorescent microscope [Axiovert 40 CFL (Carl Zeiss, Jena, Germany) or BZ-X700 (KEYENCE, Osaka, Japan)]. Cross-section area of each myofiber was measured by the ImageJ software.