Figure 4

Immunohistochemistry with TRAP, PCNA and Caspase 3. A: One sample from intermediate group showed weak positive TRAP staining (arrow) at the ossifying border of the hypertrophic chondrocytes. B. Higher magnification of black box in A, positive TRAP staining (arrow). C. No TRAP activity was detected in any of the samples from the fused group. PCNA positive cells (brown) in D. Non-deformed. Some proliferating cells can be seen at the growth zones of the vertebral body endplate (arrow). E. Higher magnification of chordoblasts in non-deformed. Positive cells were rarely found in chordoblasts. F. Intermediate vertebrae. PCNA was detected in higher amount at growth zones of the vertebral body endplate and extending abaxial and axial direction (arrow). G. Fused vertebrae. PCNA labeled cells were detected in the corresponding areas, but in higher abundance (arrow). H. PCNA positive cells were observed in the notochord and also in arch centra (arrow). I. Higher magnification of the black box in H, PCNA positive notochordal cells (arrow). Caspase 3 positive cells (black) in J. Non-deformed. Caspase 3 positive cells can be seen at the fringe of the growth zones of the vertebral body endplate (arrow). No positive cells could be detected in the chordoblasts (arrowhead) in any of the groups. K. Intermediate vertebrae; positive cells were detected in exceedingly higher amount at the corresponding areas (arrows). L. Fused vertebrae; positive cells were detected in the corresponding areas, but in higher amounts (arrow). M. Positive caspase 3 signal increased along the rims of the vertebral body and in trabeculae in fused vertebral bodies where we observed ectopic bone formation (arrow). N. Higher magnification of black box in E showing caspase 3 positive cells in the joints of ectopic bone (arrow). O. Higher magnification of caspase 3 labeled cells showing typical apoptotic phenotype with membrane blebbing (arrow). ND, non-deformed; IM, intermediated; FS, fused, nc, notochord; ns, notochordal sheath, eb, endbone; ec, ectopic bone. Scale bar = 100 μm.