Immunofluorescence analysis of EPS8 was performed using 70% confluent log phase RAW 264.7 cells treated with LPS (100 ng/mL, 24h). 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 EPS8 Polyclonal Antibody (Product # PA5-18196) at 5 microgram/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Rabbit anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A-11078) 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). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytosolic localization. Panel e represents untreated cells. Panel f shows cells with no primary antibody to assess background. The images were captured at 60X magnification.