Horseradish peroxidase conjugated anti rabbit igg

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.

Tumor tissues or cells were washed with ice-cold PBS and lysed in ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Nantong, China) to obtain whole protein extracts. The nuclear protein lysates were prepared with a NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Proteins in the lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies: polyclonal rabbit antibody against human FOXM1 (1:1000; Abcam, Cambridge, UK), rabbit anti-Cyclin D1 (1:1000; Sigma-Aldrich Co., St Louis, MO, USA), rabbit antic-Myc (1:1000; Sigma-Aldrich Co.), rabbit anti-TCF-4 (1:500; Sigma-Aldrich Co.), rabbit anti-Lamin B1 (1:1000; Abcam), and rabbit anti-β-actin (1:1000; Beyotime Institute of Biotechnology). The signals from the primary antibody were amplified by incubating with horse radish peroxidase conjugated anti-rabbit IgG (1:1000; Beyotime Institute of Biotechnology) and detected with Amersham™ ECL™ Western Blotting Detection Reagents (GE Healthcare, Boston, MA, USA).