Fig. 3. Phosphotyrosine-detection in podosomes. Chicken (A,B) or human (C) osteoclast precursors cultured in the absence (A) or presence (B,C) of RANKL-ODF were double-stained with the antiphosphotyrosine antibody 4G10 and rhodamine-phalloidin and analyzed by confocal microscopy. (B) The cell peripheries of three chicken osteoclasts, which surround an undifferentiated mononuclear cell located at the upper left side. 4G10 and rhodamine staining are depicted separately and merged (4G10, green; rhodamine phalloidin, red). Insets at the upper left of panels in A and C are examples of high power views of podosomes obtained by scanning in the z-axis to show the bright phosphotyrosine signal at podosome tips. The bottom side of these insets corresponds to the cell side which is in contact with the extracellular substrate. Panels at the far right are fivefold magnifications of image regions marked by rectangles in the adjacent panels. Merged channels (upper half) and the 4G10 channel alone (bottom half) are represented. Arrows in the right-hand panels indicate cases where strongly rhodamine-stained structures did not co-distribute with a strong phosphotyrosine staining. Arrowheads indicate co-distribution of strong phosphotyrosine and strong F-actin signals. Scale bars: 10 µm in A,B; 2 µm in insets.