Following the optical imaging session at 48 h, animals were humanely euthanized and tumor tissue was excised and used for a dye extraction protocol [11 (link)], [12 (link)]. Tumors were cut into smaller pieces and rinsed with saline. These pieces were mixed with 1 mL of radioimmunoprecipitation assay buffer and transferred to 2 mL tubes containing ceramic beads. The buffer had 50 mM Tris-base (pH 7.4), 150 mM sodium chloride (NaCl), 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) as previously described [11 (link)]. The tubes then underwent high force homogenization (Bead Mill 4 Homogenizer, Fisher Scientific, Waltham, MA) for IR-780 dye extraction. After centrifugation at 2000 g for 10 min (repeated twice), the supernatants were transferred to a 96-well black plate (200 μL per well). Controls of known IR-780 concentration were processed along with the supernatants of the tissue samples. Each tissue sample was measured in triplicate. The fluorescent signal from each well was quantified by a microplate reader (Synergy H4, BioTek, Winooski, VT) with optical excitation and emission set at 780 nm and 820 nm, respectively. NIR dye accumulation in the tumor tissue was calculated and represented as the percentage of dye retained in the tumor relative to the total dye injected into the animal.