Dab substrate solution

Mice brains were put into 4% paraformaldehyde for at least 24 h at 4°C. The brains were embedded in paraffin after dehydration and clearance. The paraffin-embedded tissue blocks were cut into sections at 4 μm thickness on a microtome. Sections on glass slides were deparaffinized and rehydrated before immunohistochemistry. Sections were boiled in citrate buffer (pH 6.0) for 20 min for antigen retrieval. The endogenous peroxidase was blocked by incubating in 3% H₂O₂ solution for 10 min at room temperature. The primary antibody c-Fos (Bioss, China) was incubated with the sections over night at 4°C. After washing, the biotinylated secondary antibody and streptavidin-HRP were sequentially incubated with the sections. After washing, the sections were stained using DAB substrate solution (Zsbio, China). The sections were counterstained by hematoxylin. Then, the sections were dehydrated and cleared before mounting coverslips. Finally, sections were visualized under a microscope at × 400 or × 100 (Olympus, Japan).

Paraffin sections of xenograft tumors were baked in an oven at 65 °C for 1 h. Then, the paraffin sections were deparaffinized in xylene and rehydrated by graded ethanol. After performing antigen retrieval with EDTA antigen retrieval solution, the sections were treated with 3% H₂O₂ to block the endogenous peroxidase activity, followed by incubating in blocking buffer (5% bovine serum albumin). Anti-HIF2α antibody (Abcam, catalog number: ab199, 1:100) and anti-CD34 antibody (Abcam, catalog number: ab81289, 1:2500) were added on the sections and incubated in a humidified chamber at 4 °C overnight. Then, the sections were incubated in horseradish peroxidase conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China) for 20 min at room temperature. DAB substrate solution (ZSGB-BIO, Beijing, China) was added on the sections to visualize the staining, followed by counterstaining with hematoxylin, dehydrating and mounting.