Biotinylated secondary antibody

The following reagents were obtained from Sigma (St. Louis, MO, USA); acetic acid, bicinchoninic acid solution, bovine serum albumin, copper(II) sulfate pentahydrate, Cresyl violet acetate, 3,3′-diamino-benzidine (DAB), radioimmunoprecipitation assay (RIPA), scopolamine hydrobromide, skim milk, and 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate (THA). The CREB, phospho-CREB, NGF, BDNF, beta-actin, and secondary horseradish peroxidase (HRP)-conjugated antibodies used for western blotting were obtained from Abcam (Cambridge, MA) and Santa Cruz Biotechnology (Santa Cruz, CA); the doublecortin (DCX), biotinylated secondary antibodies, and avidin-biotin peroxidase complex used for immunohistochemical staining were obtained from Abcam (Cambridge, MA), Santa Cruz Biotechnology (Santa Cruz, CA) and Vector Laboratories (Burlingame, CA).

The reagents were obtained from Sigma (St. Louis, MO, USA); 3,3′-diamino-benzidine (DAB), reduced glutathione (GSH) radioimmunoprecipitation assay (RIPA), scopolamine hydrobromide, skim milk, 9-Amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate (tacrine), 1,1,3,3-tetraethoxypropane (TEP). The other reagents were obtained from the following vendors: CREB, phospho-CREB, BDNF, beta-actin, and secondary horseradish peroxidase (HRP)-conjugated antibodies for western blotting (Abcam, Cambridge, MA; and Santa Cruz Biotechnology; Santa Cruz, CA), doublecortin (DCX), 4-hydroxynonenal (4HNE), Ki67, biotinylated secondary antibodies, and avidin-biotin peroxidase complex (Abcam, Cambridge, MA; Santa Cruz Biotechnology, Santa Cruz, CA; and Vector, Burlingame, CA) for immunohistochemical staining, thiobarbituric acid (TBA; Lancaster Co., Lancashire, England), H₂O₂, (Junsei Chemical Co., Ltd., Tokyo, Japan).

Anti-mouse TNF-α and IL-6 antibodies and biotinylated secondary antibodies were obtained from Abcam. The ELISA assay was carried out by using enzyme-linked immunosorbent assay (ELISA) kits (Abcam) according to the manufacturer’s protocol.

Primary OS tumor and healthy tissues were provided by the 920th Hospital of Joint Logistics Support Force of the Chinese People’s Liberation Army, Department of Orthopedics. Protein levels of MIF, CSNK2A2, VDAC2, and ODC1 in both tumor and para-cancerous tissues, were evaluated by IHC. Briefly, the tissues were thawed at room temperature and were subsequently boiled in sodium citrate at 80–90℃ for 25 min and incubated with sodium citrate containing 0.1% H₂O₂ in Tris-HCl for 1.5 h for rehydration and antigen retrieval. Tissue sections were then incubated with 3% H₂O₂ solution for 10 min, followed by incubation with 5% bovine serum albumin (FBS, Sigma, USA). Next, the samples were incubated with primary antibodies against MIF (1:500, Abcam, UK), CSNK2A2 (1:500, Abcam), VDAC2 (1:500, Abcam), and ODC1 (1:500, Abcam) at 4 °C overnight. The samples were then washed three times with Tris washing buffer and probed with biotinylated secondary antibodies (Abcam) and stained according to the instructions provided by the manufacturer of the commercial IHC kit (ThermoFisher Scientific, USA), followed by counterstaining and visualization with the ChemMate DAKO EnVision Detection kit (DAKO) and evaluation under light microscopy (ThermoFisher Scientific) by two experienced pathologists.

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).