Ar1071

After 4 weeks of treatment, the rabbits were anesthetized using intravenous
administration with 3% sodium pentobarbital (30 mg/kg) in an ear vein and the
animals were subsequently sacrificed via air embolism. The right femoral head
was divided into two parts along the coronal plane of the central hole, one
placed in liquid nitrogen cryopreservation and another fixed with 10%
formaldehyde solution (Sangon Biotech Co., Ltd., Shanghai, China) at 4°C for 48
h. Then the femoral head was decalcified with EDTA decalcification solution
(AR1071; Boster Biological Technology, Ltd., Wuhan, China) for 2 months and
embedded in paraffin. Sections of 5 μm thickness were made and stored in
thermostat of 37°C. Specimen sections were stained with hematoxylin and eosin
(HE) and Masson staining.
For immunohistochemical (IHC) staining, sections were deparaffinized, antigen
retrieved, blocked, and incubated with primary antibodies of OCN (1:400;
GTX13418; GeneTex, Irvine, CA, USA) and vascular endothelial growth factor
(VEGF) (1:250; BS1313R; Bioss, Beijing, China) and relevant secondary
antibodies. Then sections were stained with DAB (3,3′-diaminobenzidine) and
counterstained with hematoxylin. At last, each piece of the sections was
observed by light microscope (Olympus Corporation, Tokyo, Japan).

The protocols for animal experiments were approved by the Committee of Animal Experimental Centre at the Zhejiang Chinese Medical University. All animal experiments were performed in accordance with the standard guidelines of the Zhejiang Chinese Medical University. 4–6 weeks old BALB/c female mice were housed in a standard polypropylene cage. Mice were randomly subcutaneously injected with 4T1 cells (1,000 cells per mouse), NSCs (1,000 cells from a single clone per mouse) or SCs (a single clone per mouse). Tumor volume was calculated following the formula V=(L×W²)×0.5, where L and W mean mid-axis length and width, respectively. On the day of sacrifice, pulmonary metastasis was assessed by counting the macroscopic metastatic nodules according to our previous report.9 (link) All the metastasis related organs (lung specimens, heart and bone) were embedded in paraffin. For bone metastasis evaluation, leg bones (femora and tibiae) were decalcified with a decalcifying solution (BOSTER AR1071) for 20 days and embedded in paraffin. The incidence of bone metastasis was evaluated by the visible osteolysis or metastasis foci. Sections stained with H&E were evaluated and photographed.