Immunohistochemical Staining of MPC2 and Ki67

IHC staining was conducted following a standard procedure as previously described [16 (link)]. Tissues were embedded with paraffin and incised into 4 μm thick sections. Dewaxing the sections by xylene and hydrating by alcohol were then performed, followed by antigen retrieval via sodium citrate (Sangon Biotech, China) and endogenous peroxidase blocking by 0.3% H₂O₂. Bovine serum albumin (BSA, Sangon Biotech) was used to block nonspecific sites, and MPC2 (1 : 100, ab236584, Abcam) and Ki67 (1 : 100, 27309-1-AP, Proteintech, China) were used to incubate the slices for 8 hrs then with secondary antibody (1 : 100, SA00001-2, Proteintech, China) for 60 minutes. The DAB Substrate kit (# 8059S, CST) was used for visualization; then, nuclei were stained with hematoxylin. Further dehydration was performed with alcohol and sealing with neutral resin. IHC stainings were qualified using a grade scoring method, and it was scored from 1 to 4 as no, weak, moderate, and strong staining, respectively [17 (link), 18 (link)]. Besides, samples were divided into two groups according to their MPC2 staining: the low level group (scores 1 and 2) and the high level group (scores 2 and 3).