Immunofluorescence analysis of Mediator of DNA damage checkpoint protein 1 was performed using 70% confluent log phase HCT 116 Nocodazole treated (200 nmol/L for 16hrs). 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 MDC1 Polyclonal Antibody (Product # PA5-79652) 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 Nuclear foci localization of MDC1 on Nocodazole treatment. Panel e represents untreated cells with nuclear localization. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.