TUNEL staining

TUNEL Apoptosis Detection Kit (Alexa Fluor 488, Yeasen Biotech Co., Ltd., Shanghai, China) was applied to detect apoptosis in vivo on the basis of the manufacturer’s instructions. Briefly, the sections of tumour tissues were deparaffinized in xylene and rehydrated in PBS buffer. Each sample was added with 20 μg/mL proteinase K and incubated at 37 °C for 20 min. After being washed with PBS buffer for 3 times, the sections were incubated in 1× equilibration buffer for 30 min and then with 50 μL TUNEL reaction mixtures (34 μL ddH₂O + 10 μL 5 × Equilibration Buffer + 5 μL Alexa Fluor 488-12-dUTP Labelling Mix + 1 μL Recombinant TdT Enzyme) in a wet box for 1 h at 37 °C in the dark. Subsequently, the sections were washed with PBS buffer 3 times. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) Staining Solution (Beyotime, Shanghai, China) was added to stain the nucleus and incubated in dark for 5 min. The slides were observed by the fluorescence microscope (Nikon, Tokyo, Japan).