Hif 1α

The collected organs were embedded in paraffin, sliced, stained routinely with H&E and examined, as well as photographed using a Zeiss microscope. To quantify the lung metastatic burden, the metastatic nodules per lung were counted. For immunohistofluorescence analysis, the lung tissue sections with a thickness of 5 µm were deparaffinized and then repaired with citrate–EDTA antigen retrieval solution. Afterward, lung tissue sections were blocked using FBS containing 0.03% Triton X-100 and then incubated with primary antibodies containing 0.1% Tween 20 for 24 h at 4°C. After washing, lung tissue sections were incubated with secondary antibodies and DAPI solution. After mounting with a mounting medium, all sections were examined under a fluorescence microscope. Information on primary antibodies (1:400) was as follows: PIN1 (Proteintech, China); E-cadherin and Vimentin (CST, USA); N-cadherin, MMP2, SNAIL and HIF1-α (ImmunoWay, USA). Information on secondary antibodies was as follows: Alexa Fluor 488/594-conjugated goat anti-rabbit IgG (Invitrogen, USA).

Immunohistochemistry was performed with the Streptavidin-Biotin Complex (SABC) kit (Boster Bio, Pleasanton, CA, United States). The paraffin sections were first dewaxed and thermally repaired with a 3% citric acid repair solution. Then, 3% H₂O₂ was added dropwise and incubated at room temperature for 10 min. The sections were washed three times with PBS and blocked with goat serum at 37°C for 30 min. Then, the sections were incubated with diluted primary antibody [CCN1, hypoxia-inducible factor-1 α (HIF-1α), VEGF, caspase-3, Bcl-2, IL-1β, and TNF-α, 1:200, ImmunoWay, Plano, TX, United States] at 4°C overnight. On the second day, the sections were washed with PBS three times. Biotinylated goat anti-rabbit IgG was dropped and incubated at 37°C for 30 min, then washed with PBS three times. The SBC-POD reagent was added dropwise to the samples and incubated at 37°C for 30 min. For negative control groups, the primary antibody was replaced with PBS. Under the microscope, 3, 3′-diaminobenzidine was applied for 50 s and counterstained with hematoxylin, sealed with neutral gum, and then observed and photographed under the microscope. Brown cells were positive for the respective antibody, and ImageJ was used to calculate the cumulative optical density of each immunohistochemical section by the double-blind method.