Micron hm 550

Tissues were fixed in 4% paraformaldehyde overnight, cryoprotected by overnight incubation in 30% sucrose, and embedded in tissue freezing medium (VWR, 25608-930). 8 µm cryosections were obtained using a cryostat (Thermo Scientific, Micron HM 550). Sections and cells were incubated in blocking medium (PBS containing 5% donkey serum, 0.2% Triton X-100) at 4 °C overnight. For immunostaining, sections were incubated with primary antibodies at 4 °C overnight and secondary antibodies for 2 h. Primary antibodies used were as follow: aYAP1 (1:200, Abcam, ab205270), Ki67 (1:200, Abcam, ab16667), γ-H2AX (1:200, Abcam, ab81299), ACTN2 (1:200, Abcam, ab137346), cardiac troponin I (1:200, Abcam, ab56357), ATP5B (ATP synthase, H⁺ transporting mitochondrial F1 complex, beta subunit; 1:200, Abcam, ab14730), MFN1 (1:200, Abcam, ab104274), DRP1 (1:200, ProteinTech, 12957-1-AP), and 4HNE (1:200, Abcam, ab46545). After washing with blocking buffer, the slices were incubated with optimal secondary antibodies with/without WGA and/or DAPI at room temperature for 2h. After washing, samples were mounted with ProLong Diamond antifade mountant (Invitrogen, 36961) and imaged using an Olympus FV1000 confocal microscope. Laser scanning confocal microscope (Olympus, FV1000) were taken to determine confocal fluorescence images. Fluorescence intensity and cell size were measured by ImageJ.

Tibialis anterior muscles from control and denervated legs were frozen in liquid nitrogen-cooled 2-methyl butane and stored at −80°C. Cross sections (12 μm) were cut using a cryostat (Micron HM 550; Thermo Scientific, Florence, KY) and sections were stained with Gomori trichrome following Engel and Cunningham (1963). ImageJ software (NIH) was used to measure total area of each section, area containing fibrotic tissue, and myocellular cross-sectional area.