Hybrid cell count application

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).

Cross-sections were made by cutting at the mid-belly of TA muscle (at the position about 3 mm from proximal end of TA muscle). After immunostaining, fluorescent images of entire cross-sections were captured with fluorescent microscope system BZ-X710 (Keyence). Image recognition and quantification were performed by using the Hybrid Cell Count Application (Keyence). First, entire cross-sectional areas of TA muscle were measured. For quantification of Myoz1-positive area, Myoz1-stained area was recognized based on the intensity of Myoz1 staining by adjusting threshold. For quantification of dystrophin-positive area, dystrophin-stained sarcolemma was first recognized based on the intensity of dystrophin staining by adjusting threshold, and then dystrophin-positive fiber area was recognized by using inversion function. After recognition of Myoz1- and dystrophin-positive areas, the misrecognized small areas were excluded by adjusting lower limit in histogram function. Finally, errors in recognition step were corrected manually, and then Myoz1- and dystrophin-positive areas were measured. Myoz1- or dystrophin-positive area was divided by entire cross-sectional area to calculate percentage of area positive for each marker. Two side unpaired t-test was used to compare two groups.

Tissues were fixed in Mildform 10 N (Fujifilm Wako, Osaka, Japan) for 16–20 h at 4 °C and then embedded in paraffin. Three micrometer-thick sections were stained with HE after deparaffinization. For immunohistochemical analysis, 3 µm-thick sections were blocked with 5% BSA/phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 2.7 mM KCl) or a Carbo-free blocking solution (for lectin staining, Vector laboratories, Newark, CA) for 1 h at 23–26 °C after deparaffinization. Next, these sections were incubated with primary antibodies or lectins for 16–20 h at 4 °C. Samples were visualized using Alexa Flour fluorescence-conjugated secondary antibodies (Thermo Fisher Scientific). Nuclei were counterstained with Vibrance Antifade Mounting Medium containing DAPI (Vector laboratories). All sections were observed using a BIOREVO BZ-X800 microscope (Keyence, Osaka, Japan).
VE-cadherin fluorescence intensity was quantified using the hybrid cell count application (Keyence). After the HEV area with positive PNAd signals was manually encircled, the fluorescence intensity of VE-cadherin was automatically quantified in the PNAd-positive area.