VLR-IR800 was generated by creating VLR-azide using the intein-EPL reaction described above. After buffer exchange to remove MESNA and excess Cys-PEG3-azide, 5 mM DBCO-IR800 (LI-COR) was added to VLR fraction. Protein was shaken at 250 rpm, 30°C for 90 min, to facilitate the bioorthogonal azide-DBCO reaction and yield VLR-IR800. For GL261 imaging experiments, VLR-IR800 (1 mg/kg ) was intravenously administered to mice and allowed to circulate for 30 min before mice were perfused via cardiac puncture. Brains were resected, fixed in 4% paraformaldehyde, bisected, and imaged using a LiCor scanner. Serial sections were cut and stained with H&E to identify the tumor margins. n = 3 per group was generated over two independent experimental days.

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