Rabbit anti laminin l9393

8μm frozen muscle tissue sections were fixed for 3 minutes in cold 100% methanol. Slides were rinsed in 1XPBS, then blocked for 45 minutes in 1XPBS with 10% fetal bovine serum. Sections were incubated overnight at 4°C with primary antibody (rabbit anti-laminin L9393 Sigma, St. Louis, MO, USA, RRID:AB_477163, diluted 1:1000 and anti-Pax7 concentrated supernatant, clone Pax7; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA, RRID:AB_2299243) diluted 1:100 in blocking solution. The following day, slides were washed 4 × 5 min at RT in 1XPBS, incubated for 1hr at RT in secondary antibodies (anti-mouse AlexaFluor594, RRID:AB_2632539, and anti-rabbit AlexaFluor488, Jackson Immunoresearch, West Grove, PA, both diluted 1:500 in 1XPBS). Slides were washed again 4 × 5 min at RT in 1XPBS and mounted with Vectashield/DAPI before imaging. For each sample, entire sections were analyzed to count the number of Pax7+ cells and laminin-positive myofibers. The percentage of Pax7+ cells/total number of laminin-positive myofibers was calculated for each sample. The percentages of Pax7+ cells/myofibers were compared between control and KS-samples using an unpaired t-test (GraphPad Prism 9).

Cardiotoxin injury in vivo was performed as previously described (27 (link)). Briefly, anesthetized WT and Kmt2d^+/β^Geo littermate male mice were injected in the belly of the right tibialis anterior (TA) with 15μg of cardiotoxin per approved institutional protocols. Injured muscles were collected at 7 days post injury and frozen in cold isopentane for histological analyses. Sequential sections were taken to locate the injured area and immunostained with anti-laminin (rabbit anti-laminin L9393 Sigma, St. Louis, MO, USA, RRID:AB_477163, diluted 1:1000) and anti-embryonic myosin heavy chain (clone F1.652 from DSHB, 1:50, RRID:AB_528358). Images were stitched together using Affinity Photo (Serif, Nottingham, UK, RRID:SCR_016951) and fiber dimensions (minimum Feret’s diameter and myofiber area) were measured using FIJI (NIH, Bethesda, MD, USA, RRID:SCR_002285).