All specimens were fixed in formalin and embedded in paraffin blocks; the blocks were then cut into 4-µm sections. All tissue sections were double stained to assess the blood vessel endothelial cell marker cluster of differentiation 31 (CD31) and the perivascular cell marker alpha smooth muscle actin (α-SMA).
For immunohistochemical (IHC) staining, we used a Polymer Double-stain System (Mo/HRP Rb/AP, Zhong Shan Gold Bridge Biotechnology Co., Ltd, Beijing, China) according to the manufacturer’s instructions. Tissue sections were incubated simultaneously with two primary antibodies: rabbit anti-human CD31 polyclonal antibody (working dilution 1:200, Abcam, Cambridge, UK) and mouse anti-human α-SMA monoclonal antibody (working dilution 1:12,000, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Sections were incubated with anti-rabbit multimer labeled with horseradish peroxidase (brown staining) for CD31 and with anti-mouse multimer labeled with alkaline phosphatase substrate (red staining) for α-SMA. The nuclear-specific hematoxylin was used for counterstaining.
For immunohistochemical (IHC) staining, we used a Polymer Double-stain System (Mo/HRP Rb/AP, Zhong Shan Gold Bridge Biotechnology Co., Ltd, Beijing, China) according to the manufacturer’s instructions. Tissue sections were incubated simultaneously with two primary antibodies: rabbit anti-human CD31 polyclonal antibody (working dilution 1:200, Abcam, Cambridge, UK) and mouse anti-human α-SMA monoclonal antibody (working dilution 1:12,000, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Sections were incubated with anti-rabbit multimer labeled with horseradish peroxidase (brown staining) for CD31 and with anti-mouse multimer labeled with alkaline phosphatase substrate (red staining) for α-SMA. The nuclear-specific hematoxylin was used for counterstaining.