Alexa fluor 488 conjugated anti mouse rabbit

Cells were fixed with 2% paraformaldehyde (PFA)/PBS (Wako), methanol (Wako), blocked with Blocking One (Nacalai) for 45 min, and subsequently incubated with primary antibodies diluted in 5% Blocking One/ PBST (Wako) at 4°C overnight. Cells were washed three times in PBS and incubated with secondary antibodies diluted in 5% Blocking One/PBST for 1 h at room temperature. DAPI (Sigma) was used to counterstain the nuclei. Samples were visualized and photographed with BZ-710X (Keyence, Osaka, Japan) or the Opera Phenix System (PerkinElmer, Waltham, MA). The antibodies used for this study were as follows: mouse anti-myosin heavy chain (pan-MHC) monoclonal (MF20; 1:500, R&D, Minneapolis, MN), mouse anti-skeletal myosin (FAST/MYH1&2), monoclonal (MY-32; 1:100, Sigma), mouse anti-Myosin-2 (MYH2) monoclonal (MABT840; 1:100, Millipore, Burlington, MA), rabbit anti-MYH3 polyclonal (1:100, Atlas antibodies, Bromma, Sweden), mouse anti-MYH7 monoclonal (A4.840; 1:200, Santa Cruz, Dallas, TX), rabbit anti-MYH8 polyclonal (1:100, Novus Biologicals, Littleton, CO), rabbit anti-sarcomeric α actinin polyclonal (1:500, Abcam, Cambridge, UK), mouse anti-dystrophin (Rod domain) monoclonal (DYS1; 1:20, Leica, Buffalo Grove, IL), Alexa Fluor 488-conjugated anti-mouse/rabbit, and Alexa Fluor 647-conjugated anti-mouse/rabbit antibodies (1:500, Invitrogen).