Immuno coat micro slides

Tumour sections mounted on immuno-coat micro slides (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) were stained for pimonidazole to assess tumour hypoxia. After rehydration, endogenous peroxidase activity was blocked for 10 min with 0.3% hydrogen peroxide. Slides were next incubated with Hypoxyprobe-1 MAb1 (HPI Inc., Burlington, BA, USA) for 30 min at 37 °C, followed by incubation with biotin-conjugated F(ab’)₂ for 15 min at 37 °C. The bound antibody complex was then visualised by incubation with streptavidin and 3,3′-diaminobenzidine tetrahydrochloride. Images of the tumour sections stained by the anti-pimonidazole antibody were captured under a microscope (Biozero BZ-8000; Keyence Co., Osaka, Japan).

Tumor sections mounted on immunocoat micro slides (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) were used for the immunohistochemical staining of pimonidazole to assess hypoxic areas in tumor tissue. After rehydration, endogenous peroxidase activity was blocked for 10 min with 0.3% hydrogen peroxide. The slides were incubated with a monoclonal antibody to pimonidazole (HPI Inc., Burlington, BA, USA) for 30 min at 37°C, followed by incubation with biotin-conjugated F(ab′)₂ for 15 min at 37°C. The bound antibody complex was then visualized by incubation with streptavidin and 3,3′-diaminobenzidine tetrahydrochloride. The images of the tumor sections stained by the anti-pimonidazole antibody were captured under a microscope (Biozero BZ-8000; Keyence Co., Osaka, Japan).