Streptavidin horseradish peroxidase complex

Immunohistochemical staining was performed on 4 mm sections from 38 paraffin‐embedded specimens. The slides were baked at 65 °C for 30 min, deparaffinized with xylene and rehydrated with ethanol. For antigen retrieval, the slides were microwave‐treated and boiled in a 0.01 m citrate buffer (pH 6.0) for 10 min at 95 °C. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. The slides were incubated with anti‐MST3 antibody (1/100; cat number 3723S; Cell Signaling Technology) overnight at 4 °C in a humidified chamber. The tissue sections were treated with biotinylated anti‐rabbit secondary antibody (Thermo Fisher Scientific), followed by further incubation with a streptavidin–horseradish peroxidase complex (Thermo Fisher Scientific). The antigen–antibody complexes were visualized using 3,3′‐diaminobenzidine, counterstained with 10% Mayer's hematoxylin, dehydrated, and mounted in Crystal Mount. The degree of immunostaining on the formalin‐fixed, paraffin‐embedded sections was reviewed and scored independently by two observers based on the proportion of positively stained cells and the intensity of the staining. If > 10% of the tumor cells or normal colon epithelial cells were stained, we defined the specimen as positive.

The 1.5-month samples and the remaining 3-month samples were decalcified in a 10% EDTA solution. Following dehydration in a series of gradient ethanol (70–100%), the decalcified samples were embedded in paraffin for sectioning. The samples were sectioned from the center of the defects, following the coronal plane of the calvarial bone. Sections 5-μm thick were stained with HE to assess bone regeneration in the defect area. The relative area of newly formed bone was measured in five randomly selected fields of vision using ImageJ.
Immunohistochemical staining of the decalcified sections was performed to evaluate the expression of OPN and OCN. Following deparaffinization and rehydration, the endogenous peroxidase activity in the prepared sections was blocked with 3% H₂O₂. Then, the sections were subjected to antigen retrieval prior to incubation with primary antibodies against OPN (Novus Biologicals, Littleton, CO, USA) and OCN (Abcam, UK). After incubation at 4 °C overnight, the sections were treated with biotinylated secondary antibodies and incubated with streptavidin–horseradish peroxidase complex (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the sections were incubated with 3,3-diaminobenzidine (DAB) and counterstained with hematoxylin. The stained sections were captured under an upright microscope (Nikon, Tokyo, Japan).