Immunofluorescence analysis of EpCAM was performed using HT-29 and HeLa 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 EpCAM Mouse Monoclonal Antibody (Product # MA5-15640) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green) in HT-29 cells. 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 of HT-29 cells, which is a positive model for EpCAM expression showing a plasma membrane localization. Panel e represents the merged image of HeLa cells, that are null for EpCAM protein expression. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.