Anti ocn

For immunohistochemical staining, after antigen retrieval, the slides were blocked with a 5% BSA solution for 1 ​hour and incubated with mouse anti-CD68 (1:100, Invitrogen, USA), anti-iNOS (1:100, Abcam, UK), anti-CD163 (1:1000, Proteintech, USA), anti-TNFα (1:100, ZenBio, China), anti-IL1β (1:100, Proteintech, USA), anti-IL10 (1:100, Proteintech, USA), anti-TGFβ1 (1:100, Proteintech, USA), anti-MMP9 (1:100, Proteintech, USA), anti-OCN (1:100, Affinity, China), anti-HIF1α (1:100, Proteintech, USA) and anti-SDHB (1:100, Proteintech, USA) at 4 ​°C overnight. Goat anti-rabbit IgG (1:100, Servicebio, China) was used as a secondary antibody and the nuclei were stained using 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Servicebio, China). Images were captured using Aperio AT2 (Leica, German).

For in vivo histocompatibility, the sliced sections of subcutaneous implantation were subjected to H&E staining and Masson’s trichrome staining to assess the tissue integration and FBR, including cell infiltration, the number of FBGC and collagenous fibrotic capsules formation of the scaffolds. For ectopic bone formation, the sliced sections of subcutaneous implantation were incubated with anti-OCN (1:100, Affinity, China) and anti-CD31 (1:200, Affinity, China) primary antibodies, followed by treatment with HRP-conjugated or Alexa Fluor 594-labelled secondary antibodies according to a standard protocol. For histological analysis of skull reconstruction, H&E and Masson’s trichrome staining were performed. To further evaluate bone formation and angiogenesis, the OCN (1:100, Affinity, China), bone morphogenetic protein type 2 (BMP-2) (1:100, Affinity, China), Runx2 (1:100, Affinity, China), VEGF (1:100, Affinity, China) and CD31 antibodies (1:200, Affinity, China) were detected via immunofluorescence or immunohistochemistry staining. Images from stained sections were obtained using a digital slide scanner (3DHISTECH, Hungary), and image analysis was performed using IPP 6.0 to quantify the expression.