Cervical tissues from patients were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. These paraffin blocks of specimens were cut into 5-µm slices. Then, the slices were deparaffinized, rehydrated, and antigen retrieval was performed using standard techniques. The slices were incubated overnight at 4 °C with primary antibodies used as follows: 1:500 for anti-Wnt3a (ab28472, Abcam, USA), 1:500 for anti-β-catenin (ab32572, Abcam), 1:1000 for anti-CBP (ab50702, Abcam), 1:1000 for anti-SOX2 (ab92494, Abcam), and 1:1000 for anti-involucrin (ab53112, Abcam). The binding of the primary antibodies was visualized using the ChemMate Detection Kit (Boster, China). The slices were lightly counterstained with Mayer's hematoxylin for 30 s.
Immunoreactivity was semiquantitatively evaluated. The immunoreactivity score was evaluated as the intensity score × proportion score. The intensity score was defined as follows: 0, negative; 1, weak; 2, moderate; or 3, strong. The proportion score was defined as follows: 0, negative; 1, <10%; 2, 11% to 50%; 3, 51% to 80%; or 4, >80% positive cells. The total score ranged from 0 to 12. Two different pathologists evaluated all the specimens in a blinded manner.
Immunoreactivity was semiquantitatively evaluated. The immunoreactivity score was evaluated as the intensity score × proportion score. The intensity score was defined as follows: 0, negative; 1, weak; 2, moderate; or 3, strong. The proportion score was defined as follows: 0, negative; 1, <10%; 2, 11% to 50%; 3, 51% to 80%; or 4, >80% positive cells. The total score ranged from 0 to 12. Two different pathologists evaluated all the specimens in a blinded manner.