Immunofluorescence analysis of Lymphocyte antigen 6A-2/6E-1 was performed using 70% confluent log phase C2C12 cells differentiated to myotubes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Ly-6A/E Monoclonal Antibody (D7) (Product # MA1-70082) at 5 ug/ml in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Product # A11006), (1:1500), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membraneous localization. Panel e represents undifferentiated cells having low signal . Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.