Fluorescent dye labeled secondary antibodies

First, monoclonal and polyclonal antibodies, including MyoG (sc-12732, 1:150, Santa Cruz), MEF2C (#5030 s, 1:200, CST), and MyHC (sc-20641, 1:150, Santa Cruz), were added to each well in every group and then incubated for 12 h at 4 °C. The incubated cells were washed with PBS 3 times for 15 min and subsequently treated with the appropriate fluorescent dye-labeled secondary antibodies (Jackson Lab, 1:500, USA) at 25 °C for 2 h. The nuclei were stained with DAPI (Molecular Probes). The images for each group were photographed under a Nikon 80i fluorescence microscope^{18 (link)}.

C2C12 myoblast differentiation was determined by immunofluorescence staining. Primary monoclonal and polyclonal antibodies against MEF2C (#5030 s, 1:200, CST), MyoG (sc-12732, 1:150, Santa Cruz), MyHC (sc-20641, 1:150, Santa Cruz), MyHC-I (NOQ, 1:200, ab234431) and MyHC-II (My32, 1:200, ab51263) were added to each well in every group and incubated for 12 h at 4 °C. The cells were washed with PBS 3 times for 15 min and incubated with appropriate fluorescent dye-labeled secondary antibodies (Jackson Lab, 1:500, USA) at 25 °C for 2 h. The nuclei were stained with DAPI (Molecular Probes). The images for each group were photographed under a Nikon 80i fluorescence microscope [16 (link)].