Enhanced endogenous peroxidase blocking buffer

For histological analysis, liver tissues embedded in paraffin were first baked at 60 °C for 1 h and then deparaffinized with xylene and anhydrous ethanol. The tissues were sequentially treated with Triton X-100, enhanced citrate antigen retrieval solution, and Enhanced Endogenous Peroxidase Blocking Buffer (Beyotime Biotechnology, China) to break the membrane, repair the antigen, and eliminate endogenous peroxidation. After blocking with QuickBlock™ for one hour, the tissues were incubated overnight at 4 °C with the primary antibody. The next day, tissues were incubated with fluorescently labeled secondary antibody for 2.5 h before staining nuclei with DAPI. Fluorescence images were acquired and analyzed using a Thunder Imager (LEICA, Germany) fast, high-resolution, inverted fluorescence imaging system.

Osteoblasts from each group were fixed on slides, fixed in 4% paraformaldehyde at room temperature for 10 min, and rinsed using the phosphate buffer (PBS). Slides were incubated with 0.1% Triton X-100 for 20 min and rinsed using PBS. Endogenous peroxidase was blocked with enhanced endogenous peroxidase blocking buffer (Beyotime, China) for 10 min. Slides were incubated in the blocking solution for 10 min (QuickBlock™ kit, Beyotime), followed by overnight incubation at 4°C in the presence of primary antibodies, ColI (ARG21965, Taiwan, China), and OCN (AF6297, Beyotime, China). Then, they were washed thrice using PBS and incubated with a secondary antibody in 1% BSA for 30 min at room temperature. Diaminobenzidine (DAB) was used as the substrate for color development. Nuclei were stained with hematoxylin to mark all cells and observed under an inverted microscope. Negative control experiment (only PBS instead of the primary antibody was added to the specimen) was conducted to ensure the specificity of immunocytochemistry staining and exclude nonspecific staining.