Immunofluorescence analysis of Ki67 was performed using 70% confluent log phase HeLa cells serum starved for 36 hours followed by serum release for 6 hrs. 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 with Ki67 Polyclonal Antibody (Product # PA1-21520) at 10 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000 dilution) 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 the serum starved cells with reduced signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.