Affinipure fab fragment

For Pax7 immunostaining, fresh sections were fixed in 4% paraformaldehyde for 20 min, permeabilized with methanol (-20°C) for 6 min, and blocked first with a solution containing 4% bovine serum albumen (BSA; Jackson) in PBS and then with goat anti-mouse AffiniPure Fab fragment (1:100; Jackson) after antigen retrieval with 100 mmol/L sodium citrate. The sections were then incubated overnight at 4°C with an anti-Pax7 antibody (1:20; Developmental Studies Hybridoma Bank). After washing with PBS, Pax7 signals were visualized by incubating with biotin-conjugated goat anti-mouse IgG₁ (1:1000; Jackson) and Cy3-conjugated streptavidin (Jackson; 1:2500). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen). For MyoD immunostaining, the fresh sections were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100/PBS (PBST) for 10 min, and blocked by incubating with 5% BSA/6% goat serum/0.2% PBST at room temperature for 2 h. Immunostaining with anti-MyoD antibody (1:50; Santa Cruz) was performed by overnight incubation at 4°C. After washing, immunoreactive proteins were visualized by incubating with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. Nuclei were stained with DAPI (Roche). The Pax7⁺ and MyoD⁺ cells in the sections (n = 10) were counted.

Frozen TA muscles were cut transversally, fixed in 4% PFA for 20 min and permeabilized with methanol for 6 min at −20 °C. Muscle sections were then blocked with a solution containing 4% BSA in PBS followed by incubation with anti-mouse AffiniPure Fab fragment (Jackson) to avoid unspecific binding. Immunostaining with primary antibodies was performed overnight at 4 °C. Secondary antibodies coupled to Alexa Fluor 488 or 594 (Molecular Probes) were used to reveal antibody binding. Nuclei were visualized by counter staining with DAPI. Cultured cells were fixed in 4% PFA, permeabilized with either 0.25% Triton (for phopho-p38 antibody) or methanol and blocked with 4% BSA in PBS before antibody incubation. Immunostaining and detection was performed as above. Primary antibodies used were: anti-laminin (Sigma, L9393; dilution 1:1,000), anti-PJA1 (Proteintech, 17687-1-AP; dilution 1:50), anti-MYOD (BD, 554130; dilution 1:50), anti-EZH2 (AC22, Cell Signaling; dilution 1:150), anti-MyHC (MF-20, DSHB; dilution 1:30 and Santa Cruz, SC-20641; dilution 1:100), anti-KI67 (BD 556003; dilution 1:1.000), anti-HA (Santa Cruz, SC-805 and SC-7392; dilution 1:50) and anti-phospho-p38 (Cell Signalling, D3F9; dilution 1:150). Images were acquired with a Leica confocal microscope and edited using the Photoshop CS4 software. Fields reported are representative of all examined fields.

In brief, slides were fixed with 4% PFA for 15 min at RT and permeabilized in ice cold menthol for 6 min at −20 °C. Heat-mediated antigen retrieval with a 0.01 M citric acid (pH 6.0) was performed for 5 min in a microwave. After 4% BBBSA (4% IgG-free BSA in PBS; Jackson, 001-000-162) blocking, the sections were further blocked with unconjugated AffiniPure Fab Fragment (1:100 in PBS; Jackson, 115-007-003) for 30 min. The biotin-conjugated anti-mouse IgG (1:500 in 4% BBBSA, Jackson, 115-065-205) and Cy3-Streptavidin (1:1250 in 4% BBBSA, Jackson, 016-160-084) were used as secondary antibodies. Primary antibodies and dilutions were used as following: mouse anti-PAX7 (1:50, DSHB), mouse anti-MyoD (1:100, Dako, M3512), mouse anti-eMyHC (1:300, Leica, NCL-MHC-d), rabbit anti-Collagen1 (1:200; Novus, NBP1-30054), and rabbit anti-laminin (1:800, Sigma-Aldrich, L9393). Masson’s trichrome staining was performed according to the manufacturer’s instructions (ScyTek Laboratories, Logan, UT). All fluorescent images were captured with a fluorescence microscope (Leica, DM 6000B). Measurements of Collagen 1 and collagen positive area were conducted by in house ImageCount software written in MATLAB (R2014b) language.