Immunofluorescence analysis of AIF was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with AIF Polyclonal Antibody (Product # PA5-19953) at 1:250 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (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). Panel c represents mitochondrial staining using MitoTracker® Red CMXRos (Product # M7512). Panel d is a merged image of Panels a, b and c clearly demonstrating co-localization of AIF with mitotracker which specifically binds to the mitochondria. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.