Pimonidazole is a 2-nitroimidazole that is reductively activated specifically in hypoxic cells and forms stable adducts with thiol groups in proteins, peptides, and amino acids. Additionally, the amount of pimonidazole that is detected is directly proportional to the level of hypoxia within tumors in vivo. The following protocol is provided for this useful tool to interrogate the levels of hypoxia in vivo. 1.Pimonidazole hydrochloride is suspended at a concentration of 30 mg/ml in 0.9% sterile saline or PBS. 2.Multiple mice are injected intravenously (tail vein) with 60 mg/kg of the pimonidazole solution. 3.Let circulate in vivo for 90 min before the mice are euthanazied with isoflurane or carbon dioxide (CO2). Note: For animal euthanasia: In compliance with proper IACUC protocols! 4. Mice are sacrificed and organs are then removed, weighed, snap-frozen, and stored in plastic scintillation vials at -80 °C. 5. The pimonidazole stain can work on paraffin-embedded tissue, frozen tissue, and cell lines. Organs are embedded in OCT, placed in a cryostat, and cut into 10 μm-thick sections. Sections are placed on microscope slides for staining.