Immunofluorescence analysis of DNMT1 was performed using 70% confluent log phase K-562 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 1 hour at room temperature. The cells were labeled with DNMT1 Polyclonal Antibody (Product # PA1-880) at 2 µg/mL 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), 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 predominant nuclear localization. Panel e represents merged image of K-562 cells treated with 5 µM Aza deoxycytidine for 3 days (medium and drug were replaced every 24 hours). The signal is significantly lower in panel e due to down-regulation of DNMT1 expression upon Aza deoxycytidine treatment. Panel f represents untreated control cells with no primary antibody to assess background. The images were captured at 60X magnification.