Immunofluorescence analysis of Extracellular superoxide dismutase [Cu-Zn] was performed using 70% confluent log phase T-47D and Hep G2 cells. 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 SOD3 Monoclonal Antibody (4GG11G6) (Product # MA5-27661) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), 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 Cytoplasmic localization in T-47D but not in Hep G2 (Panel e) which is reported to be low to negative for SOD3 expression. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.