2.6. CD31 immunohistochemistry staining

Murine vascular blood vessels were detected using species-specific CD31 immunostaining. Unstained tumor sections were processed after conventional deparaffinization and endogenous peroxidase quenching using 3% hydrogen peroxide. Tumor tissue sections were incubated with 10% normal donkey serum in PBS for 3 h at room temperature to block non-specific binding sites. Rabbit anti-mouse CD31 primary antibody (ab28364, Abcam, UK) was diluted 1: 50 in 2% donkey serum in PBS and incubated for 3 h at room temperature, followed by incubation with the secondary anti-rabbit HRP using an X-Cell-Plus HRP Detection Kit (Menarini Diagnostics, UK) for 45 min at room temperature. The reaction was revealed using a VECTOR SG Peroxidase (HRP) Substrate Kit (Vector Laboratories, US) for 10 min at room temperature. All sections were counterstained with Neutral Fast Red (Sigma Aldrich, UK).