Vectashield fluorescent mounting media

Muscle sections were fixed in 4% PFA for 10 minutes, washed in PBS, then incubated in 0.5% Triton X-100 diluted in PBS for 10 minutes. Sections were rinsed in PBS and incubated with 3% H₂O₂ for 10 minutes to block endogenous peroxidases. After being washed with PBS, muscle sections underwent heat-mediated antigen retrieval in sodium citrate (10 mM, pH 6.5) for 20 minutes at 92 °C. Sections were then washed in PBS and block in 2.5% NHS for 90 minutes. Following blocking, sections were incubated in primary antibody overnight for Rb IgG anti-HMGB1 (1:250, ab18256; Abcam) and mouse IgG2b anti-dystrophin (1:200, D8168 Sigma; Millipore Sigma) diluted in NHS. The next day, sections were washed with PBS, incubated for 75 minutes in goat anti-rabbit IgG biotinylated secondary antibody (1:1000; Jackson ImmunoResearch) diluted in NHS, washed in PBS, and then incubated for 75 minutes in streptavidin-conjugated AF594 (1:250; Invitrogen) and goat anti-mouse IgG2b AF488 (1:250, A21141; Invitrogen) diluted in PBS for 75 minutes. Sections were then rinsed in PBS, stained with DAPI (1:10,000; Invitrogen) for 10 minutes and mounted with VectaShield fluorescent mounting media (Vector Labs).

Slides that contained myoblasts from the irradiation experiments were incubated in 0.5% Triton X-100 diluted in phosphate buffered saline (PBS) for 10 minutes and then washed in PBS. Cells were then incubated in blocking reagent (2% BSA, 0.1% Triton X-100 diluted in PBS) for 1 hour. Following blocking, cells were incubated in primary antibodies overnight for rabbit IgG anti-p16 (1:250, ab108349; Abcam, Cambridge, MA) and mouse IgG1 anti-γH2AX (1:250, 05–636; Millipore Sigma, Burlington, MA) diluted in blocking reagent. The following morning, cells were washed in PBS and then incubated in goat anti-rabbit IgG biotin (1:1000, 111–065-144; Jackson ImmunoResearch, West Grove, PA) diluted in blocking reagent for 75 minutes. Cells were then washed and incubated in streptavidin-conjugated AF594 (1:250, S11227; Invitrogen, Waltham, MA) and goat anti-mouse IgG1 AF488 (1:250, A21121; Invitrogen) diluted in PBS with 0.1% Triton X-100 for 75 minutes. Cells were then washed in PBS, stained with DAPI (1:10,000, D35471; Invitrogen) for 10 minutes and mounted with VectaShield fluorescent mounting media (H1000; Vector Labs, Burlingame, CA).

Muscle sections were fixed in 4% PFA for 10 minutes, washed in PBS, then incubated in 0.5% Triton X-100 diluted in PBS for 10 minutes. Sections were then washed in PBS and block in 2.5% NHS for 90 minutes. Following blocking, sections were incubated in primary antibody overnight for Rb IgG anti-53BP1 (1:250, ab175933; Abcam), mouse IgG1 anti-γH2AX (1:250; Millipore Sigma), and mouse IgG2b anti-dystrophin (1:200, Millipore Sigma) diluted in NHS. The next day, sections were washed with PBS, incubated for 75 minutes in goat anti-rabbit IgG AF647 (1:250; Invitrogen), goat anti-mouse IgG1 AF488 (1:250; Invitrogen), and goat anti-mouse IgG2b AF594 (1:250; Invitrogen) diluted in PBS for 75 minutes. Sections were then rinsed in PBS, stained with DAPI (1:10,000; Invitrogen) for 10 minutes and mounted with VectaShield fluorescent mounting media (Vector Labs).

Cells were fixed in 4% paraformaldehyde (PFA) for 10 minutes, wash in phosphate buffer saline (PBS), and then incubated in 0.5% Triton X-100 diluted in PBS for 10 minutes. Cells were then incubated in blocking reagent (2% BSA, 0.1% Triton X-100 diluted in PBS) for 1 hour. Following blocking, cells were incubated in primary antibodies overnight for rabbit IgG anti-p16 (1:250, ab108349; Abcam, Cambridge, MA) and mouse IgG2a anti-myosin heavy chain (1:100, A4.1025; Developmental Studies Hybridoma Bank, Iowa City, IA) diluted in blocking reagent. The following morning, cells were washed in PBS and then incubated in goat anti-rabbit IgG biotin (1:1000, 111–065-144; Jackson ImmunoResearch, West Grove, PA) diluted in blocking reagent for 75 minutes. Cells were washed and incubated in streptavidin-conjugated AF594 (1:250, S11227; Invitrogen, Waltham, MA) and goat anti-mouse IgG2a AF488 (1:250, A21121; Invitrogen) diluted in PBS with 0.1% Triton X-100 for 75 minutes. Cells were then washed in PBS, stained with DAPI (1:10,000, D35471; Invitrogen) for 10 minutes and mounted with VectaShield fluorescent mounting media (H1000; Vector Labs, Burlingame, CA).

Fixed sections were rinsed in PBS and incubated with 3% H₂O₂ for 10 minutes to block endogenous peroxidases. After being washed with PBS, muscle sections underwent heat-mediated antigen retrieval in sodium citrate (10 mM, pH 6.5) for 20 min at 92 °C. Sections were then washed in PBS and block in 2.5% NHS for 90 minutes. Following blocking, sections were incubated in primary antibody overnight for mouse IgG1 anti-Pax7 (1:100; DHSB) diluted in 2.5% NHS. The next day, sections were washed with PBS, incubated for 75 minutes in goat anti-mouse IgG1 biotinylated secondary antibody (1:1000, 115–065-205; Jackson ImmunoResearch) diluted in NHS, washed in PBS, and then incubated for 75 minutes in streptavidin horseradish peroxidase (1:500, S-911, Invitrogen) diluted in PBS. Sections were washed again in PBS, then incubated for 15 minutes in TSA AF594 (1:500, B40957, Invitrogen) diluted in PBS. Sections were then washed and incubated in primary antibody overnight for mouse IgG1 anti-γH2AX (1:250; Millipore Sigma) diluted in NHS. The following morning, sections were rinsed in PBS, incubated in goat anti-mouse IgG1 AF488 (1:250; Invitrogen) diluted in PBS for 75 minutes, rinsed in PBS, stained with DAPI (1:10,000; Invitrogen) for 10 minutes and mounted with VectaShield fluorescent mounting media (Vector Labs).