Immunofluorescence analysis of IRAK4 was performed using RAW 264.7 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 1 hour at room temperature. The cells were labeled with IRAK4 Polyclonal Antibody (Product # PA5-20018) at 5 µg/mL concentration in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant 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 SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing staining in cytoplasm. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.