Immunofluorescence analysis of PrP was performed using 70% confluent log phase A549 or MCF7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Anti-PrP Monoclonal Antibody (F89/160.1.5) (Product # MA1-750) at 1:200 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) 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, 1:300). Panel d represents the merged image showing cytosolic localization. Panel e shows no staining for PrP in MCF7 which is a negative model. Panel f represents control A549 cells with no primary antibody to assess background. The images were captured at 60X magnification.