Alexa594 conjugated α bungarotoxin

C2C12 myotubes were incubated with 10 ​μg/ml Alexa594-conjugated α-bungarotoxin (Invitrogen, 1938422). The cells were then fixed in 4 ​% paraformaldehyde (PFA) for 15 ​min. Before and after fixation, the cells were washed with phosphate buffered saline (PBS) three times. For peripherin staining in addition to α-bungarotoxin staining, the cells were treated with PBS containing 2 ​% goat serum (Fujifilm Wako Pure Chemical, WDR2410) and 0.1 ​% Triton X-100 for 1 ​h. After washing with PBS, the cells were incubated with rabbit polyclonal anti-peripherin antibody (1:300, Millipore, 2972430) overnight at 4 ​°C, and with Alexa488-conjugated goat anti-rabbit IgG (1:100, Invitrogen, SH251139) for 1 ​h at room temperature. The staining was observed with an IX71 fluorescence microscope (Olympus) and analyzed by CellSens software (Olympus). The total area and the total intensity of AChR clusters were blindly evaluated using MetaMorph software (Molecular Devices).

C2C12 myoblasts were seeded on a plate coated with 0.05 μg/μl collagen I (BD Biosciences). C2C12 myoblasts were induced to differentiate into myotubes by culturing cells in DMEM and 2% horse serum for five days. After differentiation, C2C12 myotubes were treated for 12 h with purified agrin (20 ng/ml 550-AG, R&D systems) or recombinant human FGF18 (200 ng/ml C60480, PromoKine) to induce AChR clustering in the presence or absence of an inhibitor for FGFRs (10 μM SU5402, Calbiochem) or an inhibitor for MEK1 (50 μM PD98059, Cell Signaling Technology). DMSO was used to dissolve SU5402 and PD98059, and was also added to the control. Thirty min before fixation in 2% paraformaldehyde, cells were incubated with 10 μg/ml Alexa594-conjugated α-bungarotoxin (Invitrogen) for 30 min to label AChR. Fluorescent images were observed under an Olympus XL71 fluorescence microscope and analyzed with MetaMorph software (Molecular Devices).