Goat anti rabbit poly hrp

The automatic immunohistochemical staining machine (Leica, Bond III, Germany) was used for dewaxing and antigen retrieval. After five rinses with phosphate-buffered saline (PBS), tissue array was soaked in hydrogen peroxide solution, incubated at room temperature for 10 min. Then, VCAN antibody (Abcam, ab177480, USA, 1:600) was added to the tissue array, incubated at 37°C for 1 h. After five rinses with PBS, goat anti-rabbit poly-HRP (Leica, DS9800, Germany) was added to the tissue array, incubated at 37°C for 10 min. Dewaxing and antigen repair were performed again. FAP antibody (Abcam, ab207178, USA, 1:250) was added to the tissue array, incubated at 37°C for 1 h. After five rinses with PBS, goat anti-rabbit poly-HRP (Leica, DS9800, Germany) was added to the tissue array, incubated at 37°C for 10 min. The nucleus was stained with DAPI. Finally, 3DHISTECH fluorescence imaging scanner was used for scanning, and HALO platform was used for quantitative analysis of staining results. The percentage of VCAN positive cells was used to identify the expression level of VCAN protein.