Dextran amine

Dextran amine (Invitrogen, MW 40K) was conjugated with a fluorescent dye. In brief, 5 mg of Dextran amine was dissolved in 0.5 ml of PBS (pH 8.0), and 13 μl of DyLight594-NHS (at 10 mg/ml in dimethylformamide) was added to the dextran solution. The reaction was incubated with gentle agitation at room temperature for 3 h. Unreacted dye was removed by dialysis (MWCO 10K) against PBS pH 7.4 at room temperature for 3 d and the fluorescent dextran solution was stored at 4° C. DyLight594-dextran (1 mg in 0.1 ml PBS) or PEGylated liposomal doxorubicin (Doxil, 0.2 mg in 0.1 ml) were injected by tail vein in 4T1 tumor-bearing mice and tumors were harvested after 30 min and 4 h. Prior to harvesting tumors, 0.05 mg DyLight633-anti-CD31 antibody in 0.1 ml PBS was injected by tail vein and mice were sacrificed after 10 min. Excised tumors were fixed, macrosectioned, cleared, and imaged as described above.

The training and validation datasets came from two different male rhesus macaques. The former received an injection of the anterograde/bidirectional tracer Lucifer Yellow in the anterior frontal cortex (frontal pole). The latter received an injection of the anterograde/bidirectional tracer Fluorescein in the ventrolateral prefrontal cortex (vlPFC). Surgery and tissue preparation were performed at the University of Rochester Medical Center. Details of these procedures were described previously (Haber, 1988 (link); Lehman et al., 2011 (link); Safadi et al., 2018 (link)). Briefly, each monkey received an injection of a bidirectional tracer conjugated with dextran amine (40–50 nl, 10% in 0.1 M phosphate buffer, pH 7.4; Invitrogen). Twelve days after the injection, animals were perfused and their brains were postfixed overnight and cryoprotected in increasing gradients of sucrose (10, 20, and 30%). All experiments were performed in accordance with the Institute of Laboratory Animal Resources Guide for the Care and Use of Laboratory Animals and approved by the University of Rochester Committee on Animal Resources.