FIG 2.
Detection of IRS-1 nuclear structures in glioblastoma multiforme xenografts. Human GBM12 cells were injected into the brains of immunodeficient mice (1 × 105 cells in 2 μl of PBS) by using a Hamilton syringe and a stereotactic frame. The cells were allowed to form brain tumors for 2 weeks, and tumor-bearing mice were subsequently treated with aldoxorubicin (aldoxo) (24 mg/kg of body weight by tail vein injection) (23). Seven days after treatment, mice were sacrificed, and brain tumors were extracted, formalin fixed, paraffin embedded, and sectioned. (A to C) Immunofluorescence detection of IRS-1 (anti-IRS-1 rabbit polyclonal antibody, catalog no. 06-248; Millipore) in brain tumor sections from aldoxorubicin-treated (A) and control vehicle-treated (B) mice and in tumor-free brain tissue from an aldoxorubicin-treated mouse (C). (D) Frequency of tumor cells positive for IRS-1 nuclear structures. The images were evaluated by using high-magnification confocal microscopy (original magnification, ×100). Three tumors per group were evaluated, in which 100 randomly selected fields per tumor were examined (n = 3). Data represent average values ± standard deviations. (E) High-magnification image of a single tumor cell from an aldoxorubicin-treated mouse in which anti-IRS-1 antibody recognized the ringlike structure. The same cell is also visualized by Nomarski contrast, and nuclei are labeled with DAPI (blue fluorescence). The rectangle indicates an IRS-1-positive nuclear structure, and the arrow points to the three-dimensional reconstruction of the IRS-1 ringlike structure. The image was acquired by using an FV1000 confocal microscope (Olympus), and the 3-D surface reconstruction was generated by using SlideBook 5 software (Intelligent Imaging Innovations).