Comprehensive Immunohistochemical Analysis of Tumor Microenvironment

The deparaffinized tissue sections at 4 μm thick were heated for antigen retrieval at 95 °C in citric acid buffer (pH 6.0). After treating with 3% H₂O₂, the sections were blocked with 5% goat normal serum, and incubated with primary antibody against α-SMA (1:100, ab5694, Abcam), CD31 (1:50, ab9498, Abcam, UK) and YAP1 (1:200, sc-398182, Santa Cruz, USA), separately. Microvessel density (MVD) was assessed by CD31 staining as described previously 33 (link), 34 (link). α-SMA⁺ CAFs and CD31⁺ endothelial cells were scored by mean optical density (density/area) using Image-pro plus 6.0 software. YAP1 staining in stromal tissues was scored into 5 intensities: 0, no staining; 1+, 1%-25%; 2+, 26%-50%; 3+, 51%-75%; 4+, 76%-100%. For immunofluorescence, cells were grown on pre-prepared coverslips for 24 h. After being fixed with 4% paraformaldehyde, treated by 0.1% triton-100, and incubated with 5% goat serum, the cells were separately stained with antibody specifically against FN (1:150, ab32419, Abcam), α-SMA (1:150), FAP (1:150, ab53066, Abcam), cytokeratin (CK8+CK18) (1:200, ab53280, Abcam), and CD31 (1:80) at 4 °C for overnight, then labelled with FITC-labeled secondary antibody (ZSBIO, China). The nuclei were stained with DAPI and the images were captured by a Nikon Eclipse 80i microscope.