Immunohistochemical Analysis of GSK3β in Osteosarcoma

The collected osteosarcoma tissue specimens from patients with primary metastatic osteosarcoma (n=24) and patients with nonmetastatic osteosarcoma (n=16) were fixed in 10% formalin and embedded in paraffin. The lung metastasis was diagnosed by computed tomography (CT) imaging. Expression levels of GSK3β in primary osteosarcoma tissues with or without metastasis were examined by standard immunohistochemical staining [14 (link)]. Immunohistochemical staining was carried out by the avidin-biotin method using an ABComplex/HRP kit (Waitai, China). The tissue sections were deparaffinized, antigen retrieved by microwave and blocked of nonspecific immunoreactions. Next, tissue sections were incubated with primary antibodies against GSK3β (1:100, BD Biosciences). For the negative control, primary antibodies were replaced by nonimmune mouse IgG (Abcam). After rinsing in phosphate-buffered saline, tissue sections were then incubated with biotinylated secondary antibodies (1:2000; Abcam). Subsequently, the nuclei were counterstained with hematoxylin. The quantitative analysis of GSK3β expression levels in tumor samples of patients with primary metastatic osteosarcoma and patients with nonmetastatic osteosarcoma was performed by TissueFaxs software (TissueGnostics GmbH).