3 3 diaminobenzidine (dab)

All tissue samples were fixed in 10% neutral formalin, embedded in paraffin, sectioned, dewaxed with xylene and dehydrated with gradient ethanol. Then, citric acid buffer was used for high temperature antigen repair, 3% hydrogen peroxide (Solarbio, Beijing, China) was used to block endogenous peroxidase activity, and the samples were incubated at room temperature for 20 min. Next, the slides were incubated with an anti-APEX1 primary antibody (Abcam, Cambridge, UK) at 4°C overnight. The next day, the samples were incubated with an appropriate horseradish peroxidase-conjugated secondary antibody at 37°C for 30 min. After staining with 3, 3′-diaminobenzidine (Solarbio, Beijing, China), the samples were stained with hematoxylin, dehydrated with gradient ethanol, made transparent with xylene, and sealed with neutral gum. The integrated option density (IOD) of APEX1 was chosen to determine the semiquantitative protein expression. ImageJ software (version 1.2; WS Rasband, National Institute of Health, Bethesda, MD, USA) was used to conduct deconvolution and downstream analyses.

Tissue slices and NHE1 (Affinity Biosciences, OH, USA) were incubated overnight at 4°C. The assay was performed by using an IHC detection kit (pv-0023, Beijing Bioss) in accordance with the kit's instruction manual. Positive immunoreaction was visualized by using 3,3′-diaminobenzidine (Beijing Solarbio). Images were captured with a scanning microscope (3DHISTECH, Hungary). NHE1 staining was quantitatively analyzed by using Image-Pro Plus 6 analysis software.

The tumour tissues were embedded in paraffin, sectioned, and were subjected to xylene dewaxing. Citric acid was used for antigen retrieval. A 3% H₂O₂-methanol solution was used to block endogenous peroxidase for 15 min, followed by blocking with 5% bovine serum albumin for 20 min. The primary anti-cluster of differentiation (CD)31 polyclonal antibody (1:50, ab28364, Abcam, UK) was added dropwise at 37 °C and incubated for 2 h. Goat anti-rabbit immunoglobulin G (IgG; Proteintech, USA) labelled with horseradish peroxidase was incubated at 37 °C for 30 min. 3, 3-diaminobenzidine (Solarbio, Beijing, China) was used to develop colour, and haematoxylin was used for counterstaining; the sections were then dehydrated, rendered transparent, and cover-slipped with Permount mounting medium (Thermo Fisher Scientific, Inc., USA). The sections were observed under a × 100 optical microscope (Olympus, Japan) to detect the positive protein expression in the tumour tissue.
Microvascular density (MVD) quantitative analysis was performed according to a method proposed previously (Weidner et al. 1992 ). In each section, the number of microvessels stained with CD31 was counted in five fields, and the average value was used as the MVD value of the tumour tissue.

For immunohistochemical staining analysis, the 5-μm slices were deparaffinized in xylene, hydrated with graded alcohol and then washed with 1% PBS. Subsequently, these slices were first incubated in 10 mmol/l citrate buffer (pH 6.0) at 100˚C for 10 min to retrieve the antigen and then placed in 3% hydrogen peroxide for 15 min at room temperature to block the endogenous peroxidase activity. Following rinsing with 1% PBS three times, the sections were blocked with goat serum for 30 min, incubated with rabbit polyclonal primary antibodies against the cyclooxygenase-2 (COX-2; 1:100 diluted; Wanlei Life Sciences, Shenyang, China) and inducible nitric oxide synthase (iNOS; 1:100 diluted; Wanlei Life Sciences) at 4˚C overnight. Subsequently, the sections were washed with 1% PBS, incubated with the corresponding goat anti-rabbit biotin-labeled secondary antibody (1:200; Beyotime, Shanghai, China) at 37˚C for 30 min and subsequently incubated in avidin-horseradish peroxidase complex (Beyotime). Thereafter, the sections were visualized using 3,3′-diaminobenzidine (Solarbio, Beijing, China) and counterstained with hematoxylin (Solarbio).