Histological staining was performed as previously described27 (link)
. Generally, the femoral heads were collected and washed in PBS, fixed in 4% paraformaldehyde, decalcified, dehydrated and embedded in paraffin. Sections were cut at a thickness of 5µm and stained with hematoxylin and eosin (H&E) after deparaffination. As for immunohistochemical (IHC) staining, endogenous peroxidase activity was quenched with 3% hydrogen peroxide at room temperature. Antigen retrieval was then performed with citrate buffer at 80°C for 10 minutes. Primary antibody against OCN (1:100; Beijing Biosynthesis Biotechnology Co., China) was used. The horseradish peroxidase (HRP)-conjugated secondary Goat anti-mouse antibody (1:100, Santa Cruz) was then added for an hour, followed by 3, 3’-diaminobenzidine tetrahydrochloride (DAKO, Glostrup, Denmark) for signal detection of OCN. Then the sections were rinsed, counterstained in hematoxylin, dehydrated, and mounted with p-xylene-bis-pyridinium bromide (DPX) permount (Sigma-Aldrich, St. Louis, MO, USA). Primary antibody was replaced with blocking solution in the negative controls. All incubation times and conditions were strictly controlled.
. Generally, the femoral heads were collected and washed in PBS, fixed in 4% paraformaldehyde, decalcified, dehydrated and embedded in paraffin. Sections were cut at a thickness of 5µm and stained with hematoxylin and eosin (H&E) after deparaffination. As for immunohistochemical (IHC) staining, endogenous peroxidase activity was quenched with 3% hydrogen peroxide at room temperature. Antigen retrieval was then performed with citrate buffer at 80°C for 10 minutes. Primary antibody against OCN (1:100; Beijing Biosynthesis Biotechnology Co., China) was used. The horseradish peroxidase (HRP)-conjugated secondary Goat anti-mouse antibody (1:100, Santa Cruz) was then added for an hour, followed by 3, 3’-diaminobenzidine tetrahydrochloride (DAKO, Glostrup, Denmark) for signal detection of OCN. Then the sections were rinsed, counterstained in hematoxylin, dehydrated, and mounted with p-xylene-bis-pyridinium bromide (DPX) permount (Sigma-Aldrich, St. Louis, MO, USA). Primary antibody was replaced with blocking solution in the negative controls. All incubation times and conditions were strictly controlled.
