Immunofluorescence analysis of CDC25B was performed using 70% confluent log phase HeLa and serum starved HeLa cells. 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 CDC25B Polyclonal Antibody (Product # PA5-17759) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at 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 of HeLa showing nuclear and cytoplasmic localization. Panel e represents HeLa cells, serum starved for 15 hours showing reduced signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.