The tumors were harvested and fixed in 4% paraformaldehyde, and then tumor samples were removed and embedded in paraffin. For Hematoxylin and Eosin and immunohistochemical staining, the sections were cut into sections measuring 4 µm in thickness. The tissue sections were then incubated with anti-HIF-1a antibodies (dilution: 1:200, Beyotime Biotechnology, Shanghai, China), anti-Glut-1 antibodies (dilution: 1:300, Beyotime Biotechnology, Shanghai, China) or anti-Ki-67 antibodies (dilution: 1:200, Beyotime Biotechnology, Shanghai, China), followed by horseradish peroxidase–conjugated antirabbit IgG (dilution: 1:200; Beyotime Biotechnology, Shanghai, China). Thereafter, DAB chromogenic kit (Beyotime Biotechnology, Shanghai, China) was used to stain and visualize the positive cells.
The data were analyzed using the Image-Pro-Plus 6.0 software (Media Cybernetics, USA). The integrated optical density (IOD) was employed to evaluate the area and intensity of the positive staining. The IOD of all positive stains was obtained from selected areas in each section, and the results were expressed as mean density = Sum (IOD)/Sum (Area). All the section under immunohistochemical staining were assessed by at least two observers.
The data were analyzed using the Image-Pro-Plus 6.0 software (Media Cybernetics, USA). The integrated optical density (IOD) was employed to evaluate the area and intensity of the positive staining. The IOD of all positive stains was obtained from selected areas in each section, and the results were expressed as mean density = Sum (IOD)/Sum (Area). All the section under immunohistochemical staining were assessed by at least two observers.