Immunostaining and Analysis

After 3 days cells were fixed with 2% paraformaldehyde by washing twice. Then the cells were treated with 0.5% Triton X-100 for 10-15 minutes to become permeable for antibody staining. Cells were incubated in 0.5 ml/well of Superblock buffer in TB to block unspecific staining. Cells expressing a skeletal muscle-specific protein, heavy chain myosin, were localized using MF20 primary antibody dissolved in PBS (1:10) and incubated overnight. Cells were washed with PBS and the incubated for 1.5 hours with a secondary antibody, peroxidase-conjufated AffiniPure (goat anti-mouse IgG + IgM (H + L) conjugated with horse radish peroxidase. The substrate consisted of Stable Peroxidase Buffer (1X) and Substrate DAB (diaminobenzamine, metal concentrate 10X CoCl, NiCl (1:10). Cells that expressed myosine were yellow as opposed to clear cells that did not express this skeletal muscle-specific protein. The results were analyzed by counting and calculating percentage of the cells that express skeletal myosin.