Immunofluorescence analysis of CD81 antigen was performed using 70% confluent log phase MCF7 cells (a), MCF7 treated with Protein transport inhibitor (PTI) (1X for 4hrs) (b), and Hep G2 treated with Protein transport inhibitor (PTI) (1X for 4hrs) (c). The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for overnight at room temperature. The cells were labeled with CD81 Polyclonal Antibody (Product # PA5-85150) at 1:100 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), (1:2000 dilution), for 45 minutes at room temperature (Panel a, b, c: Green). Nuclei (Panel d, e, f: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel d, e, f, g: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d, e represents the merged image showing peroxisomes, vesicles, ER, membrane localization. Panel c, f represents Hep G2 showing no expression. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.