HeLa tumors fixed in 4% paraformaldehyde were dehydrated and then embedded in paraffin. Next, samples were sectioned at 5-µm thickness and stained with hematoxylin and eosin (H&E). Tumor histopathological changes were captured with an optical microscope (Olympus Corporation).
For IHC, the sections were deparaffinized, hydrated at 70°C in xylene and microwaved at full power for 20 min for antigen retrieval using sodium citrate (pH 6.0). In addition, 5% BSA was used as the blocking reagent overnight at 4°C. Next, the slides were incubated with anti-HIF-1α primary antibody (1:100; cat. no. ab51608; Abcam), anti-NF-κB p65 primary antibody (1:200; cat. no. ab16502; Abcam) or anti-Ki67 primary antibody (1:200; cat. no. ab15580; Abcam) overnight at 4°C. The next day, the slides were washed three times with PBS and incubated with a goat anti-rabbit polyclonal HRP-conjugated secondary antibody (1:50; cat. no. 32260; Thermo Fisher Scientific, Inc.) for 2 h at room temperature. After diaminobenzidine staining (Invitrogen; Thermo Fisher Scientific, Inc.), images were captured with an optical microscope and analyzed using ImageJ 1.53 software (NIH). TUNEL assay by IHC was performed with DeadEnd™ Colorimetric TUNEL system (cat. no. G7360; Promega Corporation) according to the manufacturer's instructions.
For IHC, the sections were deparaffinized, hydrated at 70°C in xylene and microwaved at full power for 20 min for antigen retrieval using sodium citrate (pH 6.0). In addition, 5% BSA was used as the blocking reagent overnight at 4°C. Next, the slides were incubated with anti-HIF-1α primary antibody (1:100; cat. no. ab51608; Abcam), anti-NF-κB p65 primary antibody (1:200; cat. no. ab16502; Abcam) or anti-Ki67 primary antibody (1:200; cat. no. ab15580; Abcam) overnight at 4°C. The next day, the slides were washed three times with PBS and incubated with a goat anti-rabbit polyclonal HRP-conjugated secondary antibody (1:50; cat. no. 32260; Thermo Fisher Scientific, Inc.) for 2 h at room temperature. After diaminobenzidine staining (Invitrogen; Thermo Fisher Scientific, Inc.), images were captured with an optical microscope and analyzed using ImageJ 1.53 software (NIH). TUNEL assay by IHC was performed with DeadEnd™ Colorimetric TUNEL system (cat. no. G7360; Promega Corporation) according to the manufacturer's instructions.