PRSS contributes to cetuximab resistance in colorectal cancer

Tan, Zhaoli; Gao, Lihua; Wang, Yan; Yin, Huihui; Xi, Yongyi; Wu, Xiaojie; Shao, Yong; Qiu, Weiyi; Du, Peng; Shen, Wenlong; Fu, Ling; Jia, Ru; Zhao, Chuanhua; Zhang, Yun; Zhao, Zhihu; Sun, Zhiwei; Chen, Hongxing; Hu, Xianwen; Xu, Jianming; Wang, Youliang

After the mice were euthanized, the tumors were excised and fixed in formalin, embedded in paraffin, cut into 5-m sections, dewaxed and hydrated, and stained with hematoxylin and eosin using a standard technique. The IHC staining procedures were as follows: Tissue sections were incubated for 10 min at room temperature in 3% hydrogen peroxide to block endogenous peroxidase. Slides were blocked and incubated with the following primary antibodies: anti-EGFR (Nichirei Biosciences), pEGFR (Cell Signaling Technology), pERK (Thr 202 /Tyr 204 ) (Cell Signaling Technology), pAKT (Cell Signaling Technology), endomucin (eBioscience), and CD34 (Abcam). The slides were subsequently incubated with polymer-HRP antirabbit (Dako) or anti-mouse (Dako) secondary antibodies. Tissue staining was visualized using 4′,6-diamidino-2-phenylindole (DAPI). The slides were then counterstained with hematoxylin, dehydrated, and mounted. The coimmunofluorescence staining procedure for isolectin GS-IB4 (594) (Invitrogen) was as follows: After the same preprocessing for IHC staining as described above, slides were incubated with primary antibody against isolectin GS-IB4 (594) and subsequently incubated with the appropriate fluorescence-conjugated secondary antibodies; DAPI counterstaining was used to identify the nuclei.