Rabbit anti mouse laminin

Both 4% PFA-fixed and optimal cutting temperature–embedded (OCT-embedded) tissues were cut in 5 μm sections, incubated for 1 hour at room temperature in blocking buffer (PBS containing 10% BSA and 2% goat serum), and stained for 1 hour at room temperature with 1:200 rabbit anti–mouse laminin (MilliporeSigma) in blocking buffer diluted 1:2 in PBS. Tissues were then washed and further stained for 1 hour at room temperature with Dylight 650–labeled anti-Ly-6G (clone 1A8; Bio X Cell) and phycoerythin-labeled anti-CD41 (eBiosciences) at 1:200, as well as with AlexaFluor 488 goat anti–rabbit IgG (Molecular Probes) diluted 1:500 in blocking buffer. Finally, samples were counterstained with DAPI (MilliporeSigma) and mounted with Mowiol medium (81381, MilliporeSigma). Images were captured on a Leica SP8 microscope.

Primary antibodies used in the study were: sheep‐anti mouse UMOD, goat‐anti‐mouse KIM‐1, mouse‐anti‐human KIM‐1, goat‐anti mouse NGAL (R&D Systems Inc, Minneapolis, MN, USA); sheep‐anti‐human UMOD, rat anti‐mouse F4/80 (AbD Serotec/Bio‐Rad, Mississauga, ON, Canada); mouse anti‐mouse αSMA, rabbit anti‐human fibronectin (Sigma‐Aldrich, St. Louis, MO, USA); rabbit anti‐mouse/human/rat TRPV5 (Alomone Labs, Jerusalem, Israel); mouse anti‐human PCNA (eBioscience/Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti‐mouse/human/monkey megalin/LRP‐2 (Abcam, Toronto, ON, Canada); rabbit‐anti‐mouse Laminin (Millipore/Sigma), mouse‐anti‐mammalian acetylated α‐Tubulin (Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti‐mouse GAPDH (Cell Signaling Technology, Danvers, MA, USA).
Secondary horseradish peroxidase (HRP) conjugated anti‐IgG antibodies were: donkey anti‐sheep (Jackson ImmunoResearch Labs, West Grove, PA, USA); goat anti‐rat and horse anti‐mouse (Cell Signaling Technology); goat anti‐rabbit and donkey anti‐goat (Santa Cruz Biotechnology).
Secondary antibodies used in immunofluorescent microscopy were highly cross‐absorbed Alexa Fluor 488 or 568 conjugated antibodies (Invitrogen/ThermoFisher Scientific) for detection of mouse, rabbit, rat, sheep, and goat IgG.

Muscles were placed into 30% sucrose solution for cryoprotection before being embedded in optimal cutting temperature medium (OCT) and frozen in liquid nitrogen cooled isopentane. 10 μm sections were cut using a CM-3060S cryostat (Leica) and collected on charged glass slides. Histology was performed as previously described (Schmidt et al., 2017 (link), 2018 (link)) and included primary antibodies for rat anti-mouse CD31 (BioRad, Hercules, CA), rabbit anti-mouse dystrophin (BioRad, Hercules, CA), rabbit-anti mouse laminin (Sigma-Aldrich, St. Louis, MO), MyHC types I (BA-D5), IIa (SC-71), and IIb (BF-F3; Developmental Studies Hybridoma Bank, University of Iowa). Samples were mounted using Vectashield hard mount medium (Vector Labs) and imaged with an Evos FL auto microscope (Thermo Fisher Scientific, Waltham, MA).