Immunofluorescence analysis of Phospho-ZAP70/Syk (Tyr319, Tyr352) was performed using 70% confluent log phase Ramos cells treated with 1.5mM of Hydrogen peroxide+ 0.5mM of Sodium ortho vanadate for 5 mins. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-ZAP70/Syk (Tyr319, Tyr352) Rabbit Polyclonal Antibody (Product # PA5-17815) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ 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 cytoplasmic localization. Panel e shows untreated cells with reduced signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.