Immunofluorescence analysis of Macrophage colony-stimulating factor 1 was performed using 70% confluent log phase SK-BR-3 and Raji 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 M-CSF Recombinant Rabbit Monoclonal Antibody (SU0413) (Product # MA5-32139) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight, and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (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 SK-BR-3 but not in Raji cells which are reported to be low or negative for M-CSF expression. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.