Laminin Immunostaining and Fiber Measurement

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.