Biodistribution imaging in vivo

Antibodies were conjugated with DyLight 755 and purified using a DyLight 755 Antibody Labeling Kit (Thermo Scientific, 84538) in accordance with the manufacturer's specifications. BALB/c athymic nude mice were inoculated subcutaneously into the right thigh with SW480 (5 × 10⁶ cells per mouse) cells in 150 μl of a 1:1 mixture of PBS/Matrigel. When the mean tumour volume reached ∼200 mm³, DyLight 755-labelled antibodies (20 μg) were intravenously injected into the mice through the tail vein. Before imaging, mice were anaesthetized with 1.5–2.5% isoflurane (Piramal Critical Care). The whole body distribution profiles of antibodies were quantified by in vivo fluorescence using the IVIS Lumina XRMS Series III (Perkin Elmer) at the indicated time intervals. After the final scan, tumour tissues (T) and normal organs (N) were excised and imaged ex vivo. To reduce the effects of tissue autofluorescence background, manual spectral unmixing was performed and the fluorescence intensity of an identically sized region of interest was then quantified by radiant efficiency (photons s⁻¹ cm⁻² steradian⁻¹ μW⁻¹ cm⁻²) using Living Image software (PerkinElmer).