Hypoxia and Vasculature Labeling in CAM Tumors

CAMs bearing tumors were intravenously injected with pimonidazole hydrochloride (1.5 mg/CAM) to detect hypoxia and Lens culinaris agglutinin (LCA) (0.05 mg/CAM) to label the CAM blood vessel network [40 (link),41 (link),42 (link),43 (link),44 (link)], 30 min before harvesting tumors. Tumors removed from the CAM were placed directly in the cryopreservative embedding media OCT compound (Electron Microscopy Sciences, Hatfield, PA, USA, 62550-01), immediately frozen in dry ice-cooled isopentane and kept at −80 °C until sectioning for immunostaining. Sections were fixed with PFA 2% for 10 min at 4 °C. Blocking and staining were performed in BSA 2% with 10% goat serum. Pimonidazole was detected with mouse antibody (Hypoxyprobe, 1:200) and goat anti-mouse Alexa Fluor 488 (Invitrogen). Nuclei were stained with 1 µg/mL DAPI (4,6-diamidino-2-phenylindole, Invitrogen, Molecular Probes, Eugene, OR, USA). Stained sections were visualized using an Axioskop 2 phase-contrast/epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY, USA).