Rabbit anti hif1α

The tumor tissue sections (5 μm) were subjected to antigen retrieval, blocked with 1% BSA for 1 h, and incubated with primary antibody (rabbit anti-HIF1α, 1:500, rabbit anti-ki67, 1:100; Sigma) overnight at 4 °C and then with HRP-conjugated IgG (Boster, Wuhan, China) for 1 h. Further, the sections were exposed to DAB (Boster) for development. After counterstained with hematoxylin, the sections were dehydrated and subjected to microscopic examination after mounting.

Tissues and cells were lysed with RIPA buffer and the BCA kit was employed to determine protein concentration in the protein extract. The protein was separated and transferred onto membranes. The membranes were incubated with primary antibodies (rabbit anti-SOX17, 1:500, Abcam, Cambridge, UK; rabbit anti-HIF1α, 1:5000, Sigma). The membranes were then incubated with horseradish peroxidase-labeled anti-rabbit IgG (1:1000, Santa Cruz Biotech, CA). Immunoblots were visualized and captured using Bio-Rad ChemiDoc™ imaging system. GAPDH (rat, 1:1000, Santa Cruz Biotech, CA) was used as an internal reference, and the protein band image was analyzed by ImageJ2x software.