Immunofluorescence analysis of HIF1A was performed using 70% confluent log phase MCF7 cells treated with Cobalt chloride (150 µM for 48hrs). 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 HIF1A Polyclonal Antibody (Product # PA3-16521) at 1:200 dilution 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), (1:3000 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 nucleus localization in CoCl2 treated MCF7 cells. Panel e represents no staining observed for untreated MCF7 cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.