Immunohistochemical Analysis of FTO, SNAIL, and Ki-67

Paraffin-embedded sections (4 μm thick) were used for IHC. After deparaffinisation, hydration, and antigen retrieval at RT, sections were incubated with antibodies specific for FTO (27226-1-AP, Proteintech, 1:500), SNAIL (WL01863, Wanleibio, Shenyang, China, 1:500), or Ki-67 (D2H10, Cell Signalling Technology, Boston, MA, USA, 1:800) at 4 °C overnight. On the second day, the sections were processed as described previously [35 (link)]. Two independent pathologists scored the specimens according to the percentage of positively stained cells (0 = 0–4%; 1 = 5–25%; 2 = 26–50%; 3 = 51–75%; 4 = 76–100%) and the intensity of staining (0 = none, 1 = slight, 2 = moderate, 3 = strong). The percentage and intensity were multiplied to obtain the total score of one visual field. The IHC score was equal to the average of the total scores for each visual field.