Alexa fluor 594 anti rabbit igg

The extensor digitorum longus (EDL) muscle was split into four pieces and fixed with 4% PFA for 30 min. After washing with phosphate-buffered saline (PBS), the muscles were blocked by incubating overnight in a blocking solution consisting of 1% Triton X-100 and 4% BSA in PBS at 4 °C, followed by incubation for 1 day with anti-synaptophysin antibody (1:200; Abcam, #ab14692) diluted with the blocking solution at 4 °C with rotation. Subsequently, the muscles were incubated for 1 day with Alexa Fluor 488 α-Bungarotoxin (1:1000; Thermo Fisher, #1313422) and Alexa Fluor 594 anti-rabbit IgG (1:1000; Jackson ImmunoResearch, #711-585-152) diluted with the blocking solution at 4 °C with rotation. The stained muscles were counterstained with DAPI (Dojindo) and mounted with SlowFade Gold antifade reagent (Thermo Fisher). Z-stack images were captured using the confocal laser scanning microscope system TCS SP8 (Leica).

Fresh muscle samples were rapidly frozen in isopentane cooled with liquid nitrogen. Different mice from those used for the force measurement were used for immunohistological staining. Using a cryostat, 7 μm-thick frozen sections were prepared. Fresh frozen sections were fixed with acetone for 5 min at −20 °C and blocked with a protein block serum-free reagent (Agilent, Santa Clara, CA, USA) for 15 min. The specimens were incubated with primary antibodies at 4 °C overnight, followed by secondary staining. The primary and secondary antibodies used were anti-dystrophin (1:800; Abcam, Cambridge, UK, Cat# ab15277), anti-collagen III (1:100; Abcam, #ab7778), Alexa Fluor 488 anti-rabbit IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA, #711-545-152), and Alexa Fluor 594 anti-rabbit IgG (1:1000; Jackson ImmunoResearch, #711-585-152). Stained samples were mounted with SlowFade Diamond anti-fade reagent (Thermo Fisher, Waltham, MA, USA, S36972). Immunofluorescent images were obtained using an inverted fluorescence microscope DMI6000B (Leica, Wetzlar, Germany), BZ-X710 (Keyence, Osaka, Japan), and a confocal laser scanning microscope system TCS SP8 (Leica). Quantitative analyses of dystrophin and collagen III staining were performed using the Hybrid Cell Count application (Keyence).

Dual immunofluorescent staining was performed using antibodies specific to DAB2 (rabbit monoclonal) or αSMA (A2547; mouse monoclonal, Sigma-Aldrich, St Louis, MO, USA). Frozen segments of human UCB tissue were fixed in pre-cooled (−20 °C) methanol for 5 min and blocked in 1% BSA for 1 h. The segments were incubated with anti-DAB2 (dilution, 1/200) and αSMA (dilution, 1/1000) for 1 h at 4 °C and rinsed three times in PBS. The segments were incubated in Alexa Fluor 594 anti-rabbit IgG (711-585-152; Jackson ImmunoResearch, Philadelphia, PA, USA) and Alexa Fluor 488 anti-mouse IgG secondary antibody (715-545-150).