Immunohistochemical Analysis of Colorectal Cancer

IHC was prepared and performed as previously described.^{23 (link),26 (link)} Samples were respectively incubated with anti-(P)RR antibody^{27 (link),28 (link)} (1: 3000), anti-active β-catenin antibody (1: 1000; Cell Signaling Technology, Danvers, MA, USA, catalog #8814), or anti-c-Myc antibody (1: 50; Abcam, Eugene, OR, USA, catalog #ab39688), as primary antibody for 1 h. Staining was considered positive when brown granules were observed in cells. Rabbit serum from which the (P)RR antibody was isolated served as the negative control to stain colorectal tissues using the same protocol as the (P)RR antibody. As previously described,^{29 (link),30 (link)} the intensity of positive staining in cancer lesions was graded from 1 to 4 (1 as the weakest intensity and 4 as the strongest intensity) by four individuals, independently. The protein expression level in each cancer lesion was determined based on the average intensity score: 1 ≤ weak < 2, 2 ≤ middle ≤ 3, and 3 < strong ≤ 4.