Cleaved caspase 3

Immunostaining of paraffin sections was performed after dewaxing and rehydrating 4 μm thick sections. The sections were dewaxed twice with dimethylbenzene and placed in absolute ethyl alcohol, graded ethanol, and distilled water. Antigen retrieval was performed using microwave treatment in 0.01 M sodium citrate buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 95 °C for 15 min. For detection, the slides were incubated overnight at 4 °C with specific primary antibodies, including rabbit anti-human hypoxia-inducible factor (HIF)-1α antibody (1:100, Abclonal, Wuhan, China), vascular endothelial growth factor (VEGF) antibody (1:100, Proteintech, Wuhan, China), Bcl2 antibody (1:100, Abclonal, Wuhan, China), Bax antibody (1:100, Abcam, Cambridge, UK), Cleaved Caspase-3 (1:100, Abcam, Cambridge, UK), PI3K antibody (1:500, Abclonal, Wuhan, China), p-PI3K antibody (1:200, Abcam, Cambridge, UK), AKT antibody (1:200, Proteintech, Wuhan, China), p-AKT antibody (1:200, Proteintech, Wuhan, China), mTOR antibody (1:50, Cell Signalling Technology, Danvers, MA, USA), and p-mTOR antibody (1:200, Abcam, Cambridge, UK). The sections were then incubated with a horseradish-peroxidase-conjugated goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China), following the manufacturer’s instructions. After washing again, the colour was developed using colourants such as DAB, and the development time was controlled under a microscope. Finally, the nuclei were re-stained with haematoxylin, dehydrated, made transparent, and sealed to complete the immunohistochemical staining. Next, the nuclei were observed under a microscope. Four fields of view per section were averaged to minimise errors. Cells stained significantly more intensely than the background staining with a dark brown colour and uniform and consistent intensity were classified as positive cells. The intensity and density of positively immunostained cells from different slides in each group were analysed using ImageJ software (version 1.54; National Institutes of Health, Bethesda, MD, USA).