Hypoxia assay

Hypoxia assay was followed according to the manufacturer’s protocol (HPi-100, HPI, Inc.). Briefly, mice were injected i.p. with 60 mg/kg body weight of pimonidazole HCl in 0.9% saline. After 90 min, mice were intravenously perfused with 100 μg FITC-lectin (Vector Labs)/150 μl PBS and then transcardially perfused with 1% PFA; brain tissue was harvested and prepared for cryosectioning. Immunostaining against 1:50 dilution of MAb1 (4.3.11.3 mouse IgG₁ anti-pimonidazole monoclonal antibody; HPI, Inc.) was performed according to manufacturer’s recommendations using Cy3 donkey anti-mouse secondary antibody (Jackson ImmunoResearch). VECTASHIELD Mounting Media with DAPI (Vector Labs) was applied. Images were captured using an upright fluorescent microscope and SlideBook software.