In vivo Brain Tumor Xenograft Assay

All animal protocols were approved by the Johns Hopkins Animal Care and Use Committee. In vivo tumorigenesis and invasion of cells expressing either NHE9-GFP or NHE9 shRNA were assayed in 4- to 6-week-old nude/athymic, male mice (NCr-nu/nu, NCI) using a previously described brain tumor model⁵⁵ . Briefly, a total of 150,000 cells were injected into the right hemisphere of the mouse brains (coordinates X1.5, Y1.34, Z3.5 relative to bregma). Each experimental group had eight animals each. Animals dying before the termination of the experiment were excluded from the analysis. Mice were sacrificed 8 weeks after injection, and fixed and stained as previously described^{2 (link)}. Briefly, mice were subjected to a transcardiac perfusion using 4% paraformaldehyde as fixative. Dissected brain were postfixed overnight at 4°C in the same fixative, embedded in OCT compound (Tissue-Tek), frozen at -80°C, and sectioned. Then, cryosections were stained with an antibody against human nuclei (mouse monoclonal IgG1, 1:250, Millipore) and imaged to calculate tumor size and invasiveness by morphometric analysis using Image J (NIH). The slides were randomized, coded in blinded fashion and counted to prevent observer bias.