Rabbit anti ocn

Double immunofluorescent staining was performed using the following primary antibodies: mouse anti-NF-κB p65 (dilution ratio: 1:150; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CTSK (1:150; Abcam), rabbit anti-RANKL (1:150; Proteintech Group Inc., Rosemont, IL, USA), rabbit anti-OPG (1:150; Abcam), rabbit anti-OPN (1:200; Abcam), and rabbit anti-OCN (1:200; Abcam). The sections were first blocked using 5% bovine serum albumin and were then incubated with a specific primary antibody, followed by species-matched secondary antibodies (Donkey Anti-Rabbit IgG H&L Alexa Fluor 594 and Donkey Anti-Rabbit IgG H&L Alexa Fluor 488; Abcam), at a 1:200 dilution. All tissue sections were subjected to autofluorescence quenching (Vector TrueVIEW; Vector Laboratories, Inc.) and mounted with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Inc.) following the manufacturer’s protocol. Fluorescent images were acquired using a Leica TCS-SP8 confocal laser scanning microscope (Leica Biosystems, Wetzlar, Germany) within 48 h after mounting. The immunofluorescence expression of each sample was evaluated using the MFI; a.u.).

Four weeks after implantation, rats were sacrificed, and specimens were obtained. After fixation in 10% formalin solution for 4 days, the femurs were first decalcified in 10% ethylenediamine tetraacetic acid (EDTA) (Sigma) solution for 21 days. Using a cutting unit (Leica), 3.5 µm sections were obtained. Sections were processed for standard immunofluorescent histochemical staining and analysis by using primary antibodies, goat anti‐Runx2 (an endothelial cell marker; R&D Systems) and rabbit anti‐OCN (an osteoprogenitor marker; Abcam, Cambridge, England), as well as Alex 594‐conjugated secondary antibodies to corresponding species (Millipore, Billerica, MA). Cell nuclei were stained with 40, 60‐diamidino‐2‐phenylindole (DAPI, Sigma). After being air dried and coverslipped, the sections were observed under a confocal laser scanning microscope (FV‐1000, Olympus, Tokyo, Japan) with the appropriate laser beams and filter settings, and confocal images were captured. For quantitative analysis of the osteoblasts around the implants, a ROI was defined as a ring around the screw extending 200 µm from the lowest point of the thread grooves and barring the region of the screw threads. The area ratio of the total number of Runx2+ and OCN in the ROI was determined with ImageJ software. Four nonadjacent slices of each sample were analysed, with five samples in each group.

The dewaxed slide was treated with Proteinase K (Sigma-Aldrich) for proteolytic digestion and 3% H₂O₂ for the elimination of endogenous peroxidase activity. After blocking with normal goat serum, the slides were incubated with primary antibody overnight at 4 °C, respectively. The primary antibodies used in this study include: rabbit anti-OCN (Abcam), rabbit anti-IL-8 (Abcam), rabbit anti-CCL5 (Abcam), rabbit anti-IL-1β (Abcam), rabbit anti-IL-1ra (Abcam), anti-CD68 (Abcam), rabbit anti-Phospho-Histone H3S10 (Abcam), and rabbit anti-TRPM7 (Abcam). The slides were then incubated with goat anti-rabbit secondary antibody and visualized using Diaminobenzidine (DAB) staining kit (Santa Cruz Biotechnology, Santa Cruz, USA) following the manufacturer’s instruction. Immunofluorescent staining was done using Alexa-Fluor 488 conjugated anti-rabbit IgG or Alexa-Fluor 647 conjugated anti-mouse IgG secondary antibodies (Thermo Fisher Scientific) and Hoechst 33324 (Thermo Fisher Scientific). Immunofluorescent images were captured using an LSM 780 confocal microscopy (Zeiss, Germany)