Standard light microscopy

The immunohistochemical assay was performed as we described previously [31 (link)]. Briefly, the tumor tissue was fixed and embedded by using formalin and paraffin wax, respectively. Tumor sections were cut at a thickness of 4 μm, deparaffinized in xylene and rehydrated in a series of ethanol solutions (100%, 95% and 75%). After antigen retrieval in citrate buffer for 10 minutes at 97°C, 3% hydrogen peroxide was used to block endogenous peroxidase activity for 10 minutes, and 10% goat serum was used to bind nonspecific antigens for 60 minutes. Subsequently, the tumor sections were incubated with the primary antibody against CXCR4 overnight at 4°C. After the detection of immuno-signals using the Vectastain ABC kit (Vector Laboratories, Burlingame, California, USA), the tumor sections were incubated with DAB, counterstained with hematoxylin, dehydrated, and then analyzed under the standard light microscopy (Zeiss).

Tissues were fixed in 4% paraformaldehyde and decalcified in 10% EDTA. Paraffin sections were stained with Masson’s trichrome stain for morphological analysis. For IHC, antigens of de-paraffinized sections were retrieved by 0.05% trypsin or hyaluronidase (10 mg/ml) and treated with 3% H₂O₂. After blocking with 5% normal goat serum, tissues were incubated with primary antibodies in 4°C, overnight. The following rabbit polyclonal antibodies against mouse were used, anti-VEGF (Abcam, Cambridge, UK), anti-PECAM (Abcam), and anti-Osteocalcin (Millipore, Billerica, MA, USA). Sections were then incubated with anti-rabbit secondary antibody (Vectastain ABC system, Vector Laboratories, Servion, Switzerland) and developed with 0.1% 3, 39-diaminobenzidine. Images were captured using standard light microscopy (Zeiss, Oberkochen, Germany) and quantified using Image-Pro Plus software (Rockville, MD, USA). Data from three independent mice staining were used for statistical analysis.