Immunofluorescence analysis of Transcription factor p65 was performed using NIH/3T3 cells (serum-starved) and NIH/3T3 cells serum-starved for 24 hours, followed by 10 ng/mL mouse TNF alpha treatment for 30 minutes. 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. The cells were labeled with NFkB p65 Monoclonal Antibody (NF-12) (Product # MA1-24691) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (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 nuclear localization of p65 protein in NIH/3T3 treated cells. Panel e represents the merged image of NIH/3T3 untreated cells, that shows cytoplasmic localization of p65 protein. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.