4.18. In Vivo Imaging Using Near-Infrared Fluorescence (NIRF)

All animal studies were conducted in full compliance with the protocol approved by the Johns Hopkins University Animal Care and Use Committee. Single subcutaneous xenografts were formed in five-week old intact male athymic nude mice (Taconic Biosciences, Hudson, NY, USA) after a single injection of 3 × 10⁶ PSMA-positive PC-3 PIP cells behind a front leg and the injection of PSMA-negative PC-3 FLU cells on the opposite side [85]. Cells were inoculated subcutaneously in 100 µL of Hanks buffered saline solution. When tumors reached 4–6 mm in diameter, fluorescently labeled antibody variants (30 µg) were applied in a single dose of 200 µL into the tail vein. Mice were scanned under 2–2.5% isoflurane anesthesia in oxygen (2 L/min) at the stated time points using a Pearl Impulse imager (LI-COR Biosciences) equipped with 700 nm and 800 nm emission channels. All images were normalized to a single image in each set in Pearl Impulse software allowing for identical acquisition time and thresholding to manage comparison among time points, treatments, and animals. Immediately after the last scan, mice were euthanized using isoflurane-anesthetized cervical dislocation, the ventral side was opened, and uncovered tumors and inner organs were scanned.