Meta confocal microscope with zen software

Murine DEC (n = 4 fusions) and parent myoblast lines (snj MB^wt and MB^mdx, n = 4/cell type) were cultured on poly-L-lysin coated German glass coverslips (Corning Inc, New York, USA) placed in 6-well plates (Corning, New York, USA) to confirm dystrophin expression. At 21 days after fusion, cells were fixed with ice-cold acetone, washed, and unspecific antibody binding were blocked with 10% normal goat serum. Mouse monoclonal anti-dystrophin primary antibody (MANDYS8, 1:300, ThermoFischer, Waltham, MA, USA) and goat anti-mouse AlexaFluor-647 conjugated secondary antibody (1:400, ThermoFisher, Waltham, MA, USA) were used for dystrophin detection. Nuclei were counterstained with DAPI (Vector Laboratories, CA, USA). A Zeiss Meta confocal microscope with ZEN software (Carl Zeiss, Oberkochen, Germany) was used for fluorescence signal detection and analysis.

For histological analysis of heart muscles paraffin blocks were cut at 5-μm sections. Samples were deparaffinized and subsequently stained with hematoxylin-eosin (Abcam, ab245880, Cambridge, MA, USA) and mounted (Poly-Mount, PolySciences Inc., Warrington, PA, USA) to analyze heart muscle structure. A BX51/IX70 Microscope (Olympus, Japan) was used for imaging.
For immunofluorescence analysis, OCT embedded frozen heart samples were cut with a cryotome (ThermoFischer, Waltham, MA, USA) at 5-μm cross-sections. Samples were fixed with ice-cold acetone for 8 min. Blocking was performed with 10% normal goat serum in 1% BSA for 60 min. Dystrophin was detected using primary rabbit anti-dystrophin antibody (1:50, ab15277, Abcam, Cambridge, MA, USA) and secondary goat anti-rabbit Alexa Fluor 488-conjugated secondary antibody (1:500, A11001, Life Technologies, USA). Nuclei were counterstained with DAPI (ab104139, Abcam, Cambridge, MA, USA). A Zeiss Meta confocal microscope with ZEN software (Carl Zeiss, Oberkochen, Germany) was used for fluorescence signal detection and ImageJ for analysis. The number of dystrophin-positive muscle fibers in five standardized regions of each cross-section were counted and normalized to total number of fibers; two non-serial cross-sections were quantified for each animal group (n > 2, mean ± SD, one-way ANOVA with post-hoc Tukey test) at day 90.

To confirm myogenic differentiation, freshly fused DEC (n = 4 fusions) and parent myoblasts (snj MB^wt and MB^mdx) were cultured on German glass coverslips in serum-free Myogenic Differentiation Medium (Promocell, Heidelberg, Germany) supplemented with 10 μg/ml insulin to induce myogenic differentiation. After seven-day culture, cells were fixed with ice-cold acetone and unspecific antibody binding was blocked with 10% normal goat serum. Rabbit polyclonal anti-myosin heavy chain antibody (1:200, Abcam, Cambridge, MA, USA) was used as primary and goat anti-rabbit Alexa Fluor 647 (1:500, Molecular Probes, OR, USA) conjugated antibody was used as secondary antibody. Nuclei were counterstained with DAPI Vector Laboratories, CA, USA. A Zeiss Meta confocal microscope with ZEN software (Carl Zeiss, Oberkochen, Germany) was used for fluorescence signal detection and analysis.