Fluoromount g medium

Slides were dried for 10 min at room temperature. Muscle cryosections were encircled with a hydrophobic pen (Dako) and were incubated with PBS containing Triton 0.5% for 10 min and then washed three times with PBS. They were incubated with BSA 2% for 1 h at room temperature and then incubated overnight with a rabbit anti-laminin antibody (1:200, L9393, Sigma-Aldrich) at 4 °C in a moist chamber. Slides were washed three times with PBS and incubated with FITC-conjugated donkey anti-rabbit secondary antibody (1:200; 711–095-152, Jackson Laboratories) at 37 °C for 45 min. Sections were soaked for 10 s in Hoechst solution H 33342 (1:1000, B2261, Sigma-Aldrich) and were washed once with PBS before mounting with antifading Fluoromount G medium (FP-483331, Interchim). Slides were stored at 4 °C protected from light until picture acquisition.

Gastrocnemius muscles were quickly dissected, mounted in 9% Tragacanth gum (Sigma, G1128), frozen in liquid nitrogen-cooled isopentane, and kept at -80 °C. 10 µM cryosections were blocked with mouse on mouse (M.O.MTM) blocking reagent (MKB-2213, Vector Laboratories) before overnight incubation at 4 °C with anti-laminin (L9393, Sigma) primary antibody in DPBS buffer supplemented with 0.5% BSA (A7030, Sigma). The next day, slides were washed in DPBS and stained with anti-rabbit Far Red-Alexa Fluor 647 (711-175-152, Jackson ImmunoResearch) secondary antibody, in DPBS buffer supplemented with 0.5% BSA, for 1 h at 37 °C. After washing in DPBS, slides were mounted in Fluoromount G medium (FP-483331, Interchim, Montluçon, France). Whole muscle section images were acquired at a 10x magnification with a wide-field fluorescence video-microscope (Video Microscope Cell Observer, ZEISS, Oberkochen, Germany). Mosaics were then stitched (Zen 2.3 lite, Zeiss). For each sample, altered fibers were manually removed. Segmentation of all muscle fibers from a cryosection was performed using Cellpose^{39 (link),40 (link)}. Mean fiber cross-sectional area was obtained using a self-developed Python script using all muscle fibers from each section.