Fig. 4. Distribution of dextran in endocytic compartments. Osteoclasts cultured on dentine disks were incubated for 1 hour with TRITC-transferrin (A,B, red) before the addition of FITC-labelled dextran-3000 for 10 minutes (A,B, green). The cells were then fixed and processed for immunostaining with antibodies against the _vß₃ integrin (A-H, blue). Limited colocalization of both markers can be observed in the area of the ruffled border (A, yellow). The lateral view of the cell taken at the position indicated by the dotted white line in (A) confirms this observation because colocalization is seen only close to the pit (B, arrowhead). When the cells were labelled for 10 minutes with both markers together, the spatial separation between ruffled border and transferrin receptor became apparent. Transferrin is found mainly at the leading edge of the cell, whereas endocytosed dextran localized to the middle section of the osteoclasts (C,D, red and green, respectively). A similar picture was also observed when osteoclasts were prelabelled with DiI-LDL (E,F, red) for 30 minutes before the addition of FITC-labelled dextran for 10 minutes (E,F, green). LDL is found distributed throughout the osteoclasts and reaches the area close to the ruffled border, where colocalization with endocytosed dextran can be observed (E,F, yellow and arrowheads). When the cells were labelled for 5 minutes with both markers together and then chased for 25 minutes, the amount of colocalization was limited and localized to the cell periphery (G, yellow and arrowheads), whereas the area close to the ruffled border was devoid of colocalization because of the absence of dextran staining, which had moved towards the cell interior (H). Osteoclasts containing only endocytosed dextran but none of the other markers are indicated with a star in (A) and (E). The direction of movement of the cells is indicated with an open arrow in (B,D,F,H). The asterisks in (E) indicate the pits excavated by the osteoclasts and hence are devoid of surface label. Scale bar, 10 µm.