FIGURE 3. Expression of Gadd45β mRNA together with Runx2 protein in prehypertrophic chondrocytes and intracellular GADD45β protein in the hypertrophic chondrocytes that express Mmp-13 mRNA.

Adjacent longitudinal sections from mouse embryonic femurs or tibiae (15.5 dpc) were analyzed by immunohistochemistry and in situ hybridization to illustrate the expression of GADD45β in relation to Runx2 protein and MMP-13 mRNA in proliferating (pro), prehypertrophic (pre-hy), and hypertrophic (hyp) zones indicated in A. Antisense Gadd45β (A and B) and anti-GADD45β antibody (D) were used to detect Gadd45β mRNA, and protein and anti-Runx2 antibody (C) detected Runx2 protein in mouse femurs. Sense Gadd45β (insets, A and B), normal goat IgG (inset, C), and normal rabbit IgG (inset, D) were applied as negative controls. Tibiae at 15.5 dpc were stained with toluidine blue (T.Blue, E). GADD45β protein was detected in late stage hypertrophic chondrocytes (F and J), in which Mmp-13 mRNA was also detected using antisense MMP-13 (H and K). Normal goat IgG (G) and sense Mmp-13 (I) were applied as negative controls. Brown staining indicates immunodetectable protein, and purple staining indicates a positive signal using antisense probe. Original magnification: A, E–I, ×40; B–D, F, and G, ×200.