M1570

Muscles were permeabilized with 1% Triton X-100 in PBS overnight, blocked with 3% normal goat serum in 0.1% Triton X-100 in PBS (PBS-T) for two hours at RT, and incubated with rabbit anti-neurofilament (1:1000, no. 841001; BioLegend, San Diego, CA, USA) and mouse anti-slow myosin monoclonal antibody (1:200, no. MAB1628; Millipore, Burlington, MA, USA) or mouse anti-Myosin (Skeletal, Fast) antibody (1:300, M1570; Sigma-Aldrich, St. Louis, MO, USA) at 37°C for two days. Alexa Fluor 594-conjugated α-bungarotoxin (α-BTX, 1:500, B13423; Thermo Fisher Scientific, Waltham, MA, USA), which labels postsynaptic AChRs, and goat anti-mouse IgG Alexa Fluor 488 and anti-rabbit IgG Alexa Fluor 647 were incubated overnight at 37°C. Nuclei were stained with DAPI for one hour at RT. Individual muscles were then isolated under the microscope and mounted on a slide with Fluoromount-G (no. 00-4958-02; Thermo Fisher Scientific). Superior oblique was not analyzed because of technical difficulties with its dissection.