Immunohistochemical Staining of SOST Protein

Immunohistochemical staining was performed as previously described [23 (link), 24 (link)]. The samples were washed in PBS, fixed in 4% paraformaldehyde, decalcified, dehydrated, and embedded in paraffin. Sections were cut at a thickness of 5 μm and were stained with H&E after deparaffination. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 20 minutes at room temperature. Antigen retrieval was then performed with citrate buffer at 80°C for 10 minutes for immunohistochemistry detection. Primary antibody against SOST protein (1 : 100; sc-365797, Santa Cruz, CA, USA) was used. Donkey anti-goat IgG horseradish peroxidase- (HRP-) conjugated secondary antibody was then added for an hour, followed by 3,3′ diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark) in the presence of H₂O₂ for signal detection of SOST. Afterward, the sections were rinsed, counterstained in hematoxylin, dehydrated with graded ethanol and xylene, 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. The sections were examined under light microscopy (DMRXA2, Leica Microsystems Wetzlar GmbH, Germany).