Cytoskeletal organization of normal and WASp-/- osteoclasts on bone slices. Bone marrow osteoclasts were derived on bone slices. At day 10 after plating, cell cultures were fixed with 3% paraformaldehyde and permeabilized with 0.05% Triton X-100 and vinculin (A, D, G, J), and actin filaments (B, E, H, K) were detected by immunofluorescence as described in “Materials and methods.” Merged images are shown in panels C, F, I, and L. In normal cells, podosomes were assembled either in noncircular clusters across the cell body (A-C) or, more commonly, in actin rings, where they were more diffuse than on glass coverslips (D-F). Vinculin colocalized with podosomes (A-C) and actin rings (D-F). WASp-null cells were devoid of podosomes (G-I) or assembled large plaques of actin (J-L). Vinculin staining was poor in WASp-/- osteoclasts, where actin filaments were almost undetectable (G-I) and distributed throughout the cytoplasm of cells containing actin plaques (J-L). The images are representative of the cytoskeletal organization of osteoclasts detected in 3 independent experiments. Bar, 20 μm.