Immunofluorescence analysis of Synaptosomal-associated protein 25 was performed using 70% confluent log phase PC-12 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 SNAP25 Polyclonal Antibody (Product # PA5-19701) at 0.8 µg/mL 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 conjugate (Product # A27034), (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). Panel d represents the merged image showing enhanced cytoplasmic and membrane localization of SNAP-25 in PC-12 cells differentiated to neuronal phenotype with NGF (200 ng/mL 5 days). Panel e represents the undifferentiated PC-12 cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.