Immunofluorescence analysis of RRM1 (Ribonucleoside-diphosphate reductase large subunit) was performed using 80% confluent log phase U-2 OS untreated and etoposide (60uM for 48 hr) treated 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 45 minutes at room temperature. The cells were labeled with RRM1 Polyclonal Antibody (Product # PA5-17023) 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), (1:2500), 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 granular, cytoplasmic localization for RRM1 in untreated U-2 OS cells that was downregulated upon etoposide treatment (Panel e). Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.