Immunofluorescence analysis of P-Selectin was performed using HEL 92.1.7 and Raji 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 P-selectin Rabbit Monoclonal Antibody (Product # MA5-31177) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti- Rabbit IgG (Heavy Chain) 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 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 composite image showing membrane localization of P-Selectin in HEL 92.1.7 but not in Raji (Panel e). Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification..