Immunohistochemistry

Tumor xenografts were fixed in 4% neutral formalin at room temperature for 48 h, soaked in 4% paraffin, and were cut into 4-µm-thick sections. Xylene was applied for deparaffinising. Next, rehydration was implemented with an ethanol gradient. After culture with 0.3% H₂O₂ for 30 min and blocking with 5% bovine serum albumin (R&D Systems) for 45 min at 37°C, the slides were incubated all night with E2F6 (product code ab53061; 1:1,000 dilution) or Ki-67 (product code ab15580; 1:1,000 dilution; both from Abcam) at 4°C, followed by 45 min of treatment at room temperature with a horseradish peroxidase-conjugated secondary antibody (cat. no. ab205718; Abcam; 1:500 dilution). Then, 3,3′-diaminobenzidine (DAB) color reagent was applied to detect the antibody binding, and tumor xenografts were counterstained with 1% hematoxylin at room temperature for 10 min and dehydrated in ethanol. Finally, image acquisition was implemented utilizing a light microscope (×200, magnification).