Immunofluorescence analysis of Phospho-c-Raf (Ser259) was performed using 70% confluent log phase A-431 cells treated with 200 ng/mL of EGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-c-Raf (Ser259) Polyclonal Antibody (Product # 44-502) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree Celsius and then labeled with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 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 membrane localization. Panel e shows untreated cells with no signal. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.