Goat anti rat hrp

Brain tissues were harvested after euthanizing mice by cervical dislocation and fixed using 10% neutral buffered formalin (Sigma). Fixed tissues were embedded in paraffin blocks and sectioned into slices of 5 μm thickness. Consecutive sections of the brain were subjected to hematoxylin and eosin (H&E) staining for evaluation of tumor morphology and cellularity, and to bright field immunohistochemistry for markers of vascularity – CD34 (EP373Y, Abcam) and hypoxia - Carbonic anhydase IX (CAIX) enzyme (NB100–417, Novus Biologicals). After deparrafinizing and dehydrating, heat-induced epitope retrieval was performed using Bond™ Epitope Retrieval Solution (pH 9.6) for 40 min at 100°C. Slides were then incubated with the primary antibody followed by secondary antibody (goat anti-rat HRP, Invitrogen, Carlsbad, CA; 1:50) for 30 min. Bond™ Mixed DAB Refine was applied for 5 min, and rinsed with deionized water to stop the DAB reaction and counter stained with hematoxylin. Slides were finally dehydrated and mounted in synthetic mounting media. Bright field images of slides were taken using Nikon NiE: Ri2 microscope with DS-Ri2 camera using NIS elements 4.5 software. Tissue sections stained for H&E and CD34 were analyzed by board-certified pathologist. % areas of sections positively stained for CAIX were analyzed using Image J.