Anti myod antibody

ChIP assays were carried out as previously described using 150 µg of chromatin for EZH2 and MyoD, and 50 µg of chromatin for modified histones. In brief, chromatins were immunoprecipitated with anti-MyoD antibody ([sc-760] Santa Cruz Biotechnology), anti-EZH2 antibody (39901, Active Motif), anti trimethyl-histone H3K27 (07-449, Millipore), anti trimethyl-histone H3K4 (07-473, Millipore), normal rabbit (Millipore, 12-370) or normal mouse IgG (Millipore, 12-371). qPCR analysis was performed in triplicate for each of three independent experiments, using 5 ng of DNA, Go Taq qPCR Master Mix (Promega) and the following primer pairs:p57 prom

F: 5′5′ACTGAGAGC-AAGCGAACAGG-3′

R: 5′ACCTGGCTGATTGGTGATGG-3′;

Maternal p57i

F: 5′-CAGATCTGACCTCAGACCCA-3′

R: 5′-CCTGTTCCTCGCCGTCCC-3′;

Paternal p57i

F: 5′CAGATCTGACCTCAGACCCG-3′

R: 5′-GACCTGTTCCTCGCCATCCT-3′;

β-globin prom

F: 5′-GAAGCCTGATTCCGTAGAGC-3′

R: 5′-CAACTGATCCTACCTCACCTTATATGC-3′;

β-Actin prom

F: 5′-GTGACATCCACACCCAGAGG-3′

R: 5′-GAATAGCCTCCGCCCTTG-3′;

Amylase prom

F: 5′-TCAGATGGGAGGACTGCTATTGT-3′

R: 5′-GCTCACATTCCTTGGCAATATCA-3′;

Albumin prom

F: 5′-AATCGTCTTTGAGGCACCAG-3′

R: 5′-GCTCAATCTTCCCAAACAGG-3′;

Timm prom

F: 5′-ACGGATGTGGCCCTTCTGGCT-3′

R: 5′-CCGCTGCGAAACGCCCACAA-3′;

p57i

F: 5′-AACTTCCAGCAGGATGTGCC-3′

R: 5′-CATCCACTGCAGACGACCAG-3′

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.

C2C12 and C57 cells grown on glass coverslips were fixed in 4% paraformaldehyde at 4 °C for 20 min, blocked in 4% (w/v) bovine serum albumin (BSA) PBS and incubated with primary anti-sclerostin (ab63097, Abcam, Cambridge, United Kingdom) anti-MyoD antibody (Santa Cruz Biotechnology, Dallas, USA) overnight at 4 °C. Coverslips were next washed with PBS and incubated with fluorescence-labeled secondary antibodies (Thermo Scientific, Waltham, MA, USA) for 1 h at room temperature. Slides were mounted with a 10% DABCO (1,4-diazabicyclo[2.2.2]octane) solution and were observed using a Nikon A1 confocal laser scanning microscope. The confocal serial sections were processed with ImageJ software to obtain three-dimensional projections and image rendering was performed using Adobe Photoshop CS 8.0 software (Adobe Systems, San Jose, CA, USA). All the images shown in this paper are representative of at least 3 independent experiments carried out under the same conditions.