Orthotopic glioblastoma xenografts

All experimental procedures involving mice were authorized by the National Animal Experiment Board. U87MG and U373MG cells expressing either firefly luciferase and NTN1FH or NTN1(II)FH were implanted into the brain of BALB/C NU/NU athymic mice. Five mice per cell-line were used. Intracranial implantation of the cells was performed as previously published [18, 24]. Shortly, cells were trypsinized, counted and suspended into PBS. 150 000 cells in 5 μl were loaded into the Hamilton needle. The mice were anaesthetized using combination of ketamine and xylazine. Skin on mouse scalp was opened with sagittal incision and a hole was drilled into the mouse scull. The hole was positioned 2 mm right and 1 mm anterior to the bregma suture. Using stereotactic device and the Hamilton needle was inserted 3 mm deep into the mouse brain. After 1 min the needle was lifted 0,5 mm. The cells were injected into the brain during 2 min and allowed to stabilize for 1 min. After injection the Hamilton needle was slowly removed, the injection hole on the skull was covered with bone wax and the wound closed with a stitch. The recovery of the mice was enhanced by administering Temgesic (RB Pharmaceuticals) 0,3 μg/mouse on the operation day and on two following days.

The tumor growth was followed with bioluminescence imaging. 3 mg of D-Luciferin (Regis Technologies) in PBS was administered subcutaneously to each mouse and allowed to circulate for 15 min. The mice were anaesthetized with isoflurane and the emitted photons were measured using Perkin-Elmer IVIS 100 imaging system. In addition, the weight of the mice was monitored regularly to evaluate their physical condition. The mice were sacrificed 21 days after U87MG implantations and 52 days after U373MG implantation.

After sacrificing the mice their brain was immediately collected and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 min. The tissue was then washed with PBS and incubated with 30% sucrose for 24 h. After that the tissue was embedded into OCT compound (Sakura Bioteh) in a cryomold (Sakura Biotech) and frozen using liquid nitrogen. The frozen tissue blocks were stored in −80 °C. For immunofluorescence staining, 7 μm thick sections were prepared and transferred onto SuperFrost⁺ slides (Menzel-Gläser). Slides were either processed immediately or stored in −20 °C.