Immunofluorescence analysis of Trefoil factor 3 was performed using 70% confluent log phase LoVo 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 45 minutes at room temperature. The cells were labeled with TFF3 Polyclonal Antibody (Product # PA5-21081) at 10 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 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 dilution). Panel d represents the merged image showing cytosolic localization. Panel e represents HT-29 cells showing no expression of TFF3. Panel f represents control cells with no primary antibody to assess the background. The images were captured at 60X magnification.