Immunohistochemistry

For ex vivo analysis of tumors, a group of 3 animals per treatment arm were dosed for the first week cycle and were sacrificed following the final MR session. Tumors were harvested and fixed in 10% neutral buffered formalin for a minimum of 48 hours. Tumors were then sectioned and embedded in paraffin followed by cutting sections (5-μm) onto glass slides. Paraffin was removed in xylene, and slides rehydrated using gradually decreasing alcohol concentrations at 2 min/step before ending in tap water (100% ethanol, 95% ethanol, 70% ethanol, water). Slides were then stained using H&E to evaluate cell viability, a Ki-67 antibody to evaluate for cell proliferation and ApopTag to quantify apoptosis after antigen retrieval with Diva (Biocare, Concord, CA) using the Avidin/Biotin Complex System (Vectastain; Vector Labs, Burlingame, CA) and discolored with the DAB solution (Vector Labs).

Capture of images was accomplished for both non-treated and treated brain tumor sections using a digital camera interfaced with an Olympus microscope. All images were obtained at the same magnification. Because of the heterogeneous nature of the tumors, Ki-67 and apoptosis indices were quantified using the highest staining area. Briefly, staining was checked under low magnification, and then the highest staining area was identified from which images were obtained at 80x magnification. Image analysis was accomplished using ImageJ to quantify Ki-67–positive and ApopTag-positive cells and total cell numbers.