2.1. Synthesis and Characterization of Near-Infrared Fluorescence Dye-Labeled aPD-L1

The synthesis and characterization of near-infrared fluorescent dye-labeled aPD-L1 was performed in accordance with our previously reported procedure [24] with modifications. We obtained the infrared dye 800CW (IRDye 800CW) with an NHS ester from LI-COR Biosciences (Lincoln, NE, USA). The IRDye 800-aPD-L1 conjugation was performed according to the manufacturer’s instructions for high-molecular-weight protein labeling. Briefly, the pH of aPD-L1 was adjusted to above seven using 1 M potassium phosphate (pH 9). The aPD-L1 and dye were mixed at 2:1 dye:aPD-L1 mol/mol, while the mixture was protected from light via incubation at room temperature for a minimum period of 2 h. After incubation, a 1xPBS pre-equilibrated desalting column (7000 MWCO, Thermo Scientific, Waltham, MA, USA) was used to purify the labeled aPD-L1. The final product of 800CW-aPD-L1 was kept at 4 °C for further applications. A NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the amount of protein conjugated to the dye at absorbances of 280 nm (A280) and 780 nm (A780) according to the manufacturer’s instructions (LI-COR Bioscience).