ΔCD PSGL-1 distributes normally to microvilli, lipid rafts, and uropods of leukocytes. (A) PSGL-1 on surfaces of WT and ΔCD leukocytes was visualized by immunogold labeling and scanning electron microscopy. Shown are pseudo-colored images of backscattered electrons (gold detection), secondary electrons (cell surface detection), and an overlay. Arrowheads mark gold particles on microvilli. Bar represents 250 nm. Quantification of gold particles on microvilli and cell bodies of WT and ΔCD leukocytes is shown on the right. (B) WT or ΔCD leukocytes were incubated in control buffer or buffer containing methyl-β-cyclodextrin (MβCD) and then lysed in cold 1% Triton X-100. The lysate was centrifuged in a discontinuous OptiPrep gradient. Fractions collected from the top were analyzed by Western blotting with antibodies to the indicated proteins. Results are representative of 3 independent experiments. (C) WT or ΔCD neutrophils stimulated with fMLP were fixed, incubated with biotinylated rabbit anti–mouse PSGL-1 IgG, followed by streptavidin–Alexa 488. After washing, cells were incubated with goat anti–mouse CD43 IgG, followed by donkey anti–goat IgG–Alexa 546. Cells were visualized by fluorescence microscopy. The specificity of staining was confirmed with control incubations (“Immunofluorescence”). Images are representative of 40 polarized cells examined for each genotype. Bar represents 1 μm.