Immunofluorescence analysis of Myosin Skeletal Muscle was performed using C2C12 and C2C12 cells differentiated to myotubes (2% Horse serum for 5 days). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Myosin Skeletal Muscle Rabbit Polyclonal Antibody (Product # PA5-31466) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization in the differentiated C2C12 cells. C2C12 cells differentiated to Myotubes show up-regulation of Myosin Skeletal Muscle (Panel d) as compared to undifferentiated C2C12 cells (Panel e). Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.