Abc hrp solution

200 uL of a 5×10⁵ cell suspension was used to cyto-spin cells onto a glass slide. The attached cells air-dried and were fixed in 3% Paraformaldehyde and permeabilized in a 0.25% Triton-X100 in PBS solution. Endogenous peroxidase activity was blocked by incubation in a 1% H₂O₂ solution. The samples were blocked in 5% BSA in PBS, then incubated in 1∶200 Rb anti-HIF-2α (Novus) primary antibody overnight in a humidified chamber. Secondary anti-rabbit antibody was added at 1∶10,000. Horseradish peroxidase (HRP) conjugation of the antigen in ABC-HRP solution (Vector kit) was followed by rapid incubation in DAB substrate solution (Vector Kit). Slides were counterstained with hematoxylin, dehydrated and coverslip mounted. Images were captured using an Olympus CX41 microscope with a mounted Infinity2 camera and Image Capture software. Stain intensity of individual cells was quantified using Image J software [43] . The average corrected total cell staining was graphed with standard error of the mean; Student’s two-tailed T-Test was performed to calculate significance at p≤0.01.

Lung or tumor tissues from mice were fixed in 10% formalin and embedded in paraffin. Hematoxylin and eosin (HE) staining was performed using Hematoxylin and Eosin Stain Kit (Vector Labs) following the manufacturer’s protocol. For immunohistochemical staining of Ki-67, Zeb1, and CD206, tumor tissue sections were boiled with 10 mM citrate buffer (pH 6.0) for 10 min to retrieve antigen. After blocking tissues by 5% normal goat serum, anti-Ki-67 (1:500; ab15580), anti-Zeb1 (1:200; ab203829), or anti-CD206 (1:200; ab64693, all from Abcam) antibodies were applied, followed by biotin-labeled secondary antibody and ABC-HRP solution (Vector Labs). Target signals were developed using Diaminobenzidine (DAB) substrate (Vector Labs).