Myogenin

Cultured MPC-myotubes or muscle cryosections (8 μm thickness) were collected for hematoxylin and eosin (H&E) or immunostaining. To perform immunofluorescence assays, cold acetone was first used to fix the samples, followed by incubation with primary antibodies overnight at 4 °C. Antibodies used included rat anti-mouse F4/80 (1:250, eBioscience, 56-4801-80), rabbit anti-mouse IRE1α (p Ser724) (1:250, NOVUS, NB100-2323), mouse anti-mouse eIF2α (p S51) (1:250, Abcam, ab32157), mouse anti-mouse ATF6 (1:250, NOVUS, NBP1-40256), rat anti-mouse CD11b (1:250, eBioscience, 50-0112-82), rabbit anti-mouse dystrophin (1:500, Bioss, bs-14477R), rat anti-mouse laminin (1:200, Abcam, ab11576), rabbit polyclonal anti-fast myosin muscle heavy chain (MyHC, 1:500, abcam, ab91506), rabbit anti-mouse myogenin (1:500, Invitrogen, PA5-116750), rabbit anti-mouse desmin (1:500, abcam, ab32362), rabbit polyclonal anti-myosin-3 (1:500, bs-10905R, bioss), and mouse anti-mouse p38 (1:500, Santa Cruz, sc-166182). The next day, the samples were incubated with goat anti-rat IgG H&L (FITC) (1:500, Abcam, ab6840), Cy3 conjugated goat anti-rat IgG, Cy3 conjugated goat anti-rabbit IgG, Alexa Fluor 488 conjugates goat anti-mouse IgG, or Alexa Fluor 488 conjugates goat anti-rabbit IgG (1:500, Beyotime, A0507, A0516, A0428, A0423). Finally, DAPI was used for counterstaining cell nuclei. An Olympus BX51 fluorescence microscope (Olympus, Japan) was used to analyze the muscle sections or cultured MPCs. For measuring the area ratio of the target protein, more than six images were randomly selected, then the total area of positive protein in the full field of each image and the total area of each image were measured using Image-Pro Plus software (IPP, Media Cybernetics, USA). The area ratio of positive protein [(the total positive area/the total area of each image) × 100%] was calculated. For measuring the intensity of fluorescent staining, the full-image integrated optical density (IOD) and the area of interest (AOI) of all the positive stains were measured in more than six randomly selected images using IPP software. The mean fluorescence staining intensity [(IOD/AOI) × 100%] was then calculated. For measurement of the myofiber cross-sectional area (CSA), 11 randomly selected images were used. First, the total 25–50 fibers area per image (total area, pixels²) were manually evaluated and calculated by image J software (NIH, USA), then the mean myofiber CSA was calculated as the total area/fiber number.

Sections from MLD and MST of all 96 animals were cut 10 µm thick with a cryostat microtome (CM3050 S, Leica, Bensheim, Germany). The sections were used for double staining with antibodies against PAX7 (DSHB, Univ. Iowa, USA) and BrdU (#11170376001, Sigma-Aldrich, Munich, Germany). Sections were fixed in 4% paraformaldehyde solution in phosphate-buffered saline (PBS) for 20 min and subsequently washed 2 × 5 min with PBS and permeabilized with 0.1% TritonX-100 (Sigma-Aldrich, Munich, Germany) in PBS (PBST) for 10 min. Nonspecific bindings of the secondary antibody were blocked with 10% normal goat serum (NGS) in PBST for 15 min, at room temperature (RT). Slides were incubated with the primary antibody against PAX7 (1:100 in PBST incl. 1% NGS) over night, at 4 °C, in a humidity chamber. After 3 × 10 min washing with PBST, slides were incubated with an Alexa Fluor 594 goat anti-mouse IgG (H + L) secondary antibody (1: 500 in PBST, A-11032, Thermo Fisher Scientific, Schwerte, Germany) for 45 min, at room temperature, in the dark. The slides were then incubated with 2N HCl, at 37 °C, for 60 min to denature DNA and thereafter washed 3 × 5 min with PBST. Slides were incubated overnight, at 4 °C, with the antibody against BrdU (1:100 in PBST incl. 1% NGS) in a humidity chamber. After washing 3 × 10 min with PBST, slides were incubated with the secondary antibody (Alexa Fluor 488 goat anti-mouse IgG1, 1: 1000 in PBST, A-21121, Thermo Fisher Scientific, Schwerte, Germany) for 45 min, at RT, in the dark and then washed 1 × 5 min with PBST, 2 × 5 min with PBS and 1 × 10 min with distilled water and covered with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, Schwerte, Germany) and coverslips (Roth, Karlsruhe, Germany). The secondary antibody Alexa Fluor 488 goat anti-mouse IgG1, which was used to detect the BrdU primary antibody, was also able to detect the PAX7 antibody in a single immunostaining. However, when PAX7 was detected first with Alexa Fluor 594 goat anti-mouse IgG (H + L), there was no additional staining with Alexa Fluor 488 goat anti-mouse IgG1 (see Supplementary Figure S1). Thus, specific, independent fluorescence signals of PAX7 and BrdU were generated for analysis (see Supplementary Figure S2).
For double staining with DLK1 or CD163 and BrdU, the sections were first incubated with DLK1 (1:100, #21682 Abcam, Cambridge, UK) or CD163 (1:100, MCA1853, AbD Serotec, BioRad, Munich, Germany) for 2 h, detected with Alexa Fluor 594 goat anti-rabbit IgG or Alexa Fluor 594 goat anti-mouse IgG (H + L) (1: 1000 in PBST, Thermo Fisher Scientific, Schwerte, Germany), respectively, then a shorter denaturation step with 2N HCl, at 37 °C, for 20 min was applied before overnight incubation with the BrdU-antibody (1:100). After secondary antibody incubation and washing, slides were covered with Roti Mount Fluor Care + Dapi (HP20.1, Roth, Karlsruhe, Germany) to enable detection of all nuclei. To ensure specific, independent staining of CD163 and BrdU, the binding of both secondary anti-mouse IgG antibodies to the CD163 primary antibody was tested. The Alexa Fluor 488 goat anti-mouse IgG1 was also able to detect it in a single immunostaining. Again, when CD163 was detected first with Alexa Fluor 594 goat anti-mouse IgG (H + L), there was no or very weak additional staining with Alexa Fluor 488 goat anti-mouse IgG1 (see Supplementary Figure S3), which did not disturb the quantitative analysis.
Double staining of PAX7 or myogenin with DLK1 was performed similarly. First, slides were incubated overnight with antibodies against PAX7 (1:100, #199010 Abcam, Cambridge, UK) or myogenin (1:100, #1835 Abcam, Cambridge, UK). After secondary antibody incubation with Alexa Fluor 594 goat anti-mouse IgG (1: 500 in PBST, Thermo Fisher Scientific, Schwerte, Germany), the antibody against DLK1 was incubated for 2 h and detected with Alexa Fluor 488 goat anti-rabbit IgG (1: 1000 in PBST, Thermo Fisher Scientific, Schwerte, Germany). Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich, Munich, Germany). Negative controls for detection of unspecific bindings of all secondary antibodies were performed by replacing the primary antibody with 10% NGS in PBST. No unspecific bindings of the secondary antibodies were observed (see Supplementary Figure S4).