Immunohistochemistry (IHC) staining

IHC staining was conducted to evaluate the immunoreactivity of FAK. The paraffin-embedded sections, prepared using protocols aforementioned, were dewaxed with xylene, hydrated with a descending graded ethanol series. The tissue sections were then blocked using an enhanced endogenous peroxidase blocking buffer (cat. no. P0100B; Beyotime Institute of Biotechnology) at 25˚C for 10 min. After washing twice with PBS, the sections were heated to 96-98˚C for 15 min inducing epitope retrieval with a citrate antigen retrieval solution (cat. no. P0081; Beyotime Institute of Biotechnology). The sections were rinsed with PBS and then blocked with 10% goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) at 37˚C for 30 min. The sections were then probed with rabbit anti-FAK (1:250; cat. no. ab40794; Abcam) at 4˚C for 12 h followed by HRP-conjugated goat anti-rabbit secondary antibodies at 37˚C for 1 h (1:5,000; cat. no. ab205718; Abcam). Fixed tissues were stained with DAB (cat. no. P0203; Beyotime Institute of Biotechnology) at 25˚C for 30 min. After washing thoroughly with distilled water, the sections were stained with hematoxylin at 25˚C for 10 min and then differentiated with an acidic ethanol differentiation solution at 25˚C for 5 sec (cat. no. C0107; Beijing Solarbio Science & Technology, Co., Ltd.). After further rinsing with distilled water, the sections were hydrated with a graded alcohol series and dehydrated with xylene. They were observed under x400 magnification with an inverted light microscope under white light and the images were collected using Image-Pro Plus (version 6.0; Media Cybernetics, Inc.). FAK expression was indicated as yellowish-brown particles.