Ab53778

Immunohistochemistry (IHC) was performed in paraffin-embedded sections on the basis of the standardized protocol. Briefly, paraffin-embedded sections (2–4 μm) were deparaffinized with xylene and rehydrated in series gradient ethanol (100%, 95%, and 85% for 1 min, respectively) at room temperature. Heat antigen repair was performed in an autoclave (121°C for 1.5 min) in a citrate sodium buffer (0.01 M), and then endogenous peroxidase was blocked using 3% hydrogen peroxide for 10 min. Sections were incubated with anti-human antibody SMAD6 (ab80049, Abcam), TGFB2 (ab53778, Abcam), PITX2 (ab98297, Abcam), CALML4 (A5086, Zhongshan Golden Bridge, Beijing), and NRP1 (ab25998, Abcam) for 1 h at 37°C and then incubated with a rabbit polyclonal antibody for 20 minutes. After dying with diaminobenzidine for 5–10 min at room temperature, sections were sealed with neutral balata, respectively. The sections were evaluated with semiquantitative method. Briefly, more than 400 cells were counted in each section, while some necrotic cells and peripheral-colored cells were elided. More than 10% of cells were nuclear; staining in all cells was defined as protein-positive expression, while less than 10% was protein-negative expression.

Ex vivo explants were fixed for 48 h in 4% paraformaldehyde, decalcified in 4% paraformaldehyde solution in PBS for one week, embedded in paraffin and cut in 5.0 μm thick sections. For morphological analysis of the new tissue, samples were stained with a Gomori’s trichrome kit (Leica Biosystems, Nussloch, Germany) to highlight collagen synthesis and osteoid deposition. Microscopic sections were analyzed with a BX43 microscope (Olympus Europa SE & Co, Hamburg, Germany).
Immunohistochemical staining was performed with recombinant rabbit monoclonal anti-mouse Runx-2 antibody (Abcam, ab192256, dilution 1:200), rabbit monoclonal anti-human osteopontin (Opn) (Abcam, ab63856, dilution 1:100), rabbit polyclonal anti-human TGF-β2 (Abcam, ab53778, dilution 1:100) and rabbit polyclonal anti-human decorin (DCN) IgG (Abcam, ab151988, dilution 1:200) primary antibodies. For visualization, Novocastra Peroxidase/DAB kit (Leica Biosystems, Nussloch, Germany) was utilized according to the manufacturers’ instructions.