Alexa fluor 594 conjugated α bungarotoxin

Muscle implants were fabricated as previously described^{11 (link)}. Briefly, MPC’s from F344 rats were suspended in type I oligomeric collagen (2.0 mg/mL) and flowed through a 4mm cylindrical mold. Once polymerized, implants were cultured under passive tension in Dulbecco’s Modified Eagle Medium (DMEM) (Fisher Scientific, Chicago, IL) supplemented with 1% penicillin, streptomycin, amphotericin B (PSF-1; HyClone, Logan, UT), and 10% fetal bovine serum (HyClone; Logan Utah) at 37^oC and 5% CO₂ for 5 days with medium changes every 2 days. On day 5, medium was changed to differentiation medium, representing DMEM supplemented with 8% horse serum (HyClone) and 1% PSF-1, and constructs were cultured for an additional 7 days to induce myotube formation.
For a subset of experiments, MPCs were induced to express motor endplates (MEE) as described previously^{14 (link)}. Briefly, muscle constructs were allowed to differentiate for 5 days at which point acetylcholine chloride (40 nM; Tocris Bioscience, Bristol, England), agrin (10 nM; R&D Systems, Minneapolis, Minnesota), and neuregulin (2 nM; R&D Systems) were added to the medium. Constructs were cultured an additional 7 days with medium changes every 3 days. Motor endplate expression was confirmed by immunostaining with Alexa Fluor 594 conjugated α-bungarotoxin (Molecular Probes, Eugene, Oregon).

After harvested specimens were incubated for a 12-hour period with 4% paraformaldehyde at 4°C, they were transferred into 30% sucrose at 4°C for an additional 24 hours. Axial cryosectioning was performed with 12-μm thickness on 3% gelatin-coated slides. The cryosections were washed in PBS 3 times. They were incubated in 0.3 mol/L glycine in PBS for 20 minutes. The sections were then permeabilized in 0.1% Triton X-100 in PBS for 20 minutes. They were washed with PBS 3 times and then blocked with 4% goat serum for 1 hour. After an overnight incubation at 4°C with rabbit polyclonal class III β-tubulin (Covance, Richmond, California; PRB-435p) at 1/1,000 dilution, slides were incubated with Alexa Fluor 488 conjugated goat anti-rabbit immunoglobulin G secondary antibody (Invitrogen; A11034) for 1 hour. To visualize the motor end plates in the same sections, we incubated them with Alexa Fluor 594 conjugated α-bungarotoxin (Molecular Probes; B13423; dilution 1:100) for 1 hour. The sections were observed with the Olympus Flowviewer 1,000 mpe confocal/2-photon microscope.

Muscle implants were fabricated as previously described.^{3 (link)-10 (link)} Briefly, MPCs from biopsies were suspended in type I oligomeric collagen (GeniPhys, Zionsville, IN) (2.0 mg/mL) and flowed through a 10-mm cylindrical mold. Once polymerized, implants were cultured under passive tension in DMEM supplemented with 1% PSF-1, and 10% FBS at 37 C and 5% CO2 for 2 days, with medium changes every 2 days. On day 3, medium was changed to differentiation medium, representing DMEM supplemented with 2% horse serum (HyClone) and 1% PSF-1, and constructs were cultured for an additional 5 days to induce myotube formation, at which point acetylcholine chloride (40 nM; Tocris Bioscience, Bristol, England), agrin (10 nM; R&D Systems, Minneapolis, MN), and neuregulin (2 nM; R&D Systems) were added to the medium to induce motor endplate formation. Constructs were cultured an additional 5 days with medium changes every 3 days. Motor endplate expression was confirmed by immunostaining with Alexa Fluor 594 conjugated α-bungarotoxin (Molecular Probes, Eugene, Oregon).