Immunofluorescence analysis of Angiotensinogen was performed using 70% confluent log phase Hep G2 cells treated with 1X protein transport inhibitor (Product # 00-4980-03) for 4 hours. Angiotensinogen is readily secreted out in the medium after translation and post processing. Treatment of cells with protein transport inhibitor sequesters Angiotensinogen in the endoplasmic reticulum. 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. cells were labeled with Angiotensinogen Monoclonal Antibody (Product # MA5-29010) at 1:200 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (Product # A27034) at a dilution of 1:2000 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). Panel d represents the merged image showing ER membrane/cytoplasmic localization. Panel e represents untreated HepG2 cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.