Cells were fixed with 4% paraformaldehyde (PFA) for 20 min and then permeabilized with 0.2% Triton X-100 for 5 min. After blocking with 1% bovine serum albumin (BSA) for 2 hours, the cells were incubated for 2 hours at room temperature with a primary antibody. A secondary antibody was then administered for 2 hours at room temperature. The primary antibodies were mouse anti-neuron–specific βIII tubulin (Tuj1, 1:100), mouse anti-human ChAT (1:200), rabbit anti-human islet1 (1:100), rat anti-human α-actinin (1:500), rat anti-human GFAP (1:200), rabbit anti-human P-gp (1:500), mouse anti-human ZO-1, and mouse anti-human occludin (1:500). The secondary antibodies were Alexa Fluor 555 anti-rabbit immunoglobulin G (IgG) (H+L) (1:500), Alexa Fluor 405 anti-rabbit IgG (H+L) (1:500), Alexa Fluor 488 goat anti-mouse IgG (H+L) (1:500), Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1:500), and Alexa Fluor 647 goat anti-rat IgG (H+L). F-actin was stained with Alexa Fluor 488 or 647 phalloidin (Cytoskeleton Inc., Denver, CO, USA) and DAPI for 20 min at room temperature, followed by three rinses with Dulbecco’s phosphate-buffered saline with Ca2+ and Mg2+ (DPBS++). To stain the nAChR clusters, the neuromuscular motor units were incubated with αBTX-conjugated Alexa Fluor 647 (2 mg/ml, Molecular Probes) for 1 hour at 37°C in a 5% CO2 incubator. The constructs were then rinsed with PBS, fixed for 1 hour with 4% PFA in PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and blocked with 2% BSA in PBS overnight. To detect cells in an apoptosis stage (caspase activity) in the muscle fiber bundles, they were incubated with CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min. All cells and samples were observed using a phase-contrast microscope (Axiovert 200, Zeiss, Germany) and a confocal laser scanning microscope (FV-1000, Olympus, Japan).

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