Immunohistochemical Analysis of CXCL12, Wnt5a, and β-Catenin

A Leica paraffin slicer RM2235 (Leica Biosystems, Solms, Germany) was used to cut 4 µm-thick sections from each TMA block. Sections were incubated at 70 °C for 1 h, dewaxed in xylene, and rehydrated using an ethanol gradient. Following antigen retrieval by boiling in citrate buffer for 20 min, sections were incubated in hydrogen peroxide for 10 min to quench endogenous peroxidase. Then, tissues were incubated overnight with primary polyclonal antibodies targeting CXCL12 (1:100, 3530, Cell Signaling Technology, Manassas, USA), Wnt5a (1:200, ab174963, Abcam) and β-Catenin (1:300, ab230216) at 4 °C. PBS was used as a negative control. After washing with PBS, sections were incubated with binding buffer and amplification agent (reagent A, GTVisionTM III Kit supply, Shanghai, China), stained with 3, 3'-diaminobenzidine (DAB, reagent B and C, GTVisionTM III Kit supply, Shanghai, China), and counterstained with hematoxylin. Staining was scored based on the percentage of positively-stained cells in each section (no positive staining or ≤ 5% = 0; 6%-25% = 1; 26%-50% = 2; 51%-75% = 3, and 76%-100% = 4), and the staining intensity (no staining = 0, weak staining = 1, moderate staining = 2 and strong staining = 3) 17 (link). For each case, two random fields were imaged, with one core viewed under high magnification (×200). All slides were analyzed by two pathologists in a blinded manner.