Approximately 48 hours following CED of ZsGreen BPNs, mice were euthanized and transcardially perfused with 10 ml of 2% heparinized 0.9% saline, followed by 10 ml of tris-buffered saline with calcium chloride (0.1 g/liter). Brains were removed, rapidly frozen to −80°C, and cut into 100-μm sections using a cryostat (1905, Leica, Buffalo Grove, IL). Every other section within 2 to 3 mm of the injection site was collected on a slide and mounted with permanent mounting medium (P36970, Invitrogen, Carlsbad, USA). Sections were imaged using a Nikon Eclipse TE2000 confocal microscope (Nikon, Melville, NY) under ×4 magnification. Multiple images were taken and stitched together in montages to capture the entire injection site. Volume of transfected tumor tissue was quantified from these images using a MATLAB script similar to previous studies (32). Briefly, background fluorescence was subtracted and images were thresholded at 5% of the maximum intensity. The total volume of transgene expression was calculated by multiplying the area of distribution from each slice by the slice thickness and summing values for each slice.

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