Immunofluorescence analysis of Allograft inflammatory factor 1 was performed using log phase THP-1 cells differentiated to Macrophages (PMA 100nM 48 hours). 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 IBA1 Recombinant Rabbit Monoclonal Antibody (31) (Product # MA5-29012) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 488 (Product # A32790) (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 dilution). Panel d represents the merged image showing cytoplasmic and plasma membrane-like localization. Panel e represents untreated THP-1 cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.