Immunofluorescence analysis of SLUG was performed using 70% confluent log phase MCF-7 cells treated with 10 ng/mL TGF-Beta for 4 days. The cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled Anti-SLUG Polyclonal Antibody (Product # PA5-20289) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 (Product # A27034) 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 nuclear localization. Panel e represents untreated MCF-7 cells having low expression of SLUG. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.