All animal studies were approved by IACUC at the University of Wisconsin-Madison. Nonobese diabetic–severe combined immunodeficient mice underwent intracranial implantation of U87 cells expressing luciferase as previously described (44). Tumor engraftment was determined by quantifying luminescence using luciferin and an IVIS imager, according to manufacturer’s protocols. For accumulation studies, tumors were grown until mice demonstrated tumor burden by displaying neurologic symptoms or hunched posture. For VLR-Fc studies, protein (3 mg/kg) was administered intravenously and allowed to circulate for 30 min. Mice were then perfused, and the organs were harvested. Organs were snap-frozen, embedded in OCT (optimal cutting temperature), and cut using a cryostat. Sections were stained with Isolectin GS-IB4 (1:400), Hoechst 33342 (1:800), and goat anti-rabbit AF555 (1:500) and then imaged on an upright Zeiss Imager Z2 fluorescent microscope. For doxorubicin accumulation studies, mice bearing U87 intracranial tumors received a single injection of P1C10- or RBC36-targeted doxorubicin-loaded liposomes intraperitoneally (12 mg doxorubicin/kg). Drug was allowed to circulate for 30 min, and then, mice were perfused with PBS and euthanized. Whole brains were bisected at the tumor site, and coronal images for doxorubicin fluorescence were taken using an IVIS imager (excitation, 465 nm; emission, 600 nm). Slices adjacent to the brain bisection used to expose the tumor were stained with H&E to define tumor margins and Isolectin GS-IB4 + Hoechst 33342 (1:400 and 1:800, respectively) for imaging on an upright Zeiss Imager Z2 fluorescent microscope. A minimum of three fields per group were quantified to determine the VLR channel mean pixel intensity using ImageJ.Data are presented as mean pixel intensity ± SD. For survival studies, after confirming tumor engraftment by luminescence, mice were intraperitoneally administered 12 mg/kg doxorubicin of P1C10-targeted (n = 5), 192-targeted (n = 5), or RBC36-targeted (n = 4) doxorubicin-loaded liposomes weekly for four cycles. Mice were monitored for neurological symptoms, hunched posture, and weight loss throughout the study and euthanized in accordance with guidelines of the IACUC protocol. The proportion of surviving mice over time was plotted using a Kaplan-Meier plot, and statistically significant differences were determined with log-rank tests.

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