After three-dimensional histomorphometric analysis, the calvaria samples were fixed in 4% paraformaldehyde, decalcified in 10% EDTA, washed in running water, dehydrated, diaphanization, and embedded in paraffin. Tissues were sectioned into 4 μm slices for hematoxylin and eosin (H&E) staining and immunohistochemistry of Dkk3, Runx2, and OCN. Briefly described, the samples underwent treatment in a pressure cooker for 1 min at 125°C in EDTA buffer (pH 8.0) and were then cooked for another 2 min and 30 s at 100°C. The samples were allowed to cool to room temperature before incubation with 3% hydrogen peroxide for 15 min. Subsequently, the sections were incubated overnight at 4°C with primary antibody and then incubated sequentially with secondary antibody and diaminobenzidine for 20, 20, and 10 min, respectively. Finally, the slides were counterstained with hematoxylin (Abcam), dehydrated, and mounted.
For immunofluorescence analysis, tissues were cut into 6 μm sections. Frozen sections were incubated for 14 h at 4°C with primary antibodies for Dkk3, Ki67, and β-catenin and incubated with CY3-conjugated secondary antibodies (ab6939, Abcam, MA, USA) at room temperature for 2 h. Slides were observed under a laser scanning confocal microscope (Leica, Wetzlar, Germany).
For immunofluorescence analysis, tissues were cut into 6 μm sections. Frozen sections were incubated for 14 h at 4°C with primary antibodies for Dkk3, Ki67, and β-catenin and incubated with CY3-conjugated secondary antibodies (ab6939, Abcam, MA, USA) at room temperature for 2 h. Slides were observed under a laser scanning confocal microscope (Leica, Wetzlar, Germany).