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IHC staining was performed to assess relative factors expression in OSCC patient tissue samples. After deparaffinization and rehydration, the tissue slides were heated in a water bath at 100 °C with citrate buffer solution (pH 6.0) for 20 min to retrieve antigen and then cooled at room temperature. The primary antibodies were incubated overnight at 4 °C in a humidified chamber and then visualized using a 3,3′-diaminobenzidine (DAB) detection kit (Dako, Glostrup, Denmark) containing goat secondary antibody molecules and DAB chromogen. Every step of the wash used phosphate-buffered saline solution (PBS) for 5 min and was repeated three times. The primary antibodies (with their dilutions reported), including RRAD (1:50, ab238584, Abcam, MA, USA), GLUT3 (1:1000, 20403-1-AP, Proteintech, Wuhan, China) and Ki-67 (1:1000, 28074-1-AP, Proteintech, Wuhan, China) were used according to the manufacturer’s instructions. The intensity of the RRAD and GLUT3 immunoreaction was scored as follows: 0 = negative, absence of stained cells; 1 = weak; 2 = moderate; and 3 = strong. The IHC staining score was calculated by multiplying the percentage of positive cells by the staining intensity. The scoring was conducted by researchers who were blinded to the clinical information of the patients.

Immunohistochemistry was performed as previously described [10 (link)]. Briefly, sections were incubated with the rabbit polyclonal antibody against GDF15 (1:100) (Abcam, UK) overnight at 4°C and visualized using 3,3′-diaminobenzidine (DAB) detection kit (Dako Cytomation, Denmark) containing goat secondary antibody molecules against rabbit immunoglobulin and DAB chromogen. Negative control was performed by using PBS instead of anti-GDF15 antibody. Two pathologists performed blind examination with a microscope. The GDF15 positive proportion score was the percentage ratio of positive GDF15-stained tumor cells to the total number of tumor cells, classified as: 0 (0%), 1 (1–10%), 2 (11–50%), 3 (51–80%), 4 (>80%). The GDF15 intensity score was the staining intensity by visual assessment and was scored as: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong).