Ab15277

Cells and skeletal muscle tissues were fixed with 4% paraformaldehyde in PBS for 15 min at 4°C, prior to embedding in Frozen Section Compound (Leica Microsystems) for cryosections. Fixed samples were incubated with 0.1%Triton X-100 in PBS for 5 min, BlockingOne (Nakacai Tesque) for 30 min, and anti-GFP (Molecular Probes A6455; diluted 1:500), anti-PAX3 (DSHB AB528426; diluted 1:100), anti-PAX7 (DSHB AB528428; diluted 1:100), anti-myogenin (DAKO M3559; diluted 1:100), anti-MyoD (Abcam ab64159; diluted 1:500), anti-laminin (Enzo Life Sciences ALX-804-190; diluted 1:500), anti-dystrophin (Abcam ab15277; diluted 1:200), anti-MyHC (R&D MAB4470; MF20, diluted 1:200), anti-FSP1 (Abcam ab124805; diluted 1:200), anti-lamin A/C (Abcam ab8984; diluted 1:200), and anti-Ki67 (Abcam ab15580; diluted 1:500) antibodies in 5% BlockingOne overnight at 4°C. After three washes with 0.1% Tween 20 in PBS, cells were incubated with Alexa-conjugated anti-mouse immunoglobulin G1 (IgG1), mouse IgG2b, rabbit IgG, or rat IgG antibodies (Molecular Probes; diluted 1:500). Cells were washed and mounted in ProLong Diamond antifade reagent with DAPI (Molecular Probes). Images were collected and processed by BZ-X710 microscopy.

Cells were lysed with RIPA buffer (Thermo Fisher Scientific) supplemented with cOmplete Protease Inhibitor Cocktail (Roche) and 5 mM EDTA. The total amount of lysate protein was quantified by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). DMD and myosin heavy chain (MHC) were detected using a Wes™ Simple Western system (ProteinSimple) with 1.25 µg of sample loading. The primary antibodies used were anti-Dystrophin (Abcam, ab15277, diluted 50-fold) and anti-MHC (R&D Systems, MAB4470, diluted 25-fold). The secondary antibodies used were anti-rabbit IgG HRP-linked antibody (ProteinSimple, 042–206) and anti-mouse IgG HRP-linked antibody (ProteinSimple, 042-205), respectively.