Fluorescence imaging of frozen sections of nPLTs

For nPLT penetration, Balb/c mice were subcutaneously injected with 1 × 106 4T1 cells per mouse in the right flank. When the tumors reached 200 mm3, these 4T1 tumor–bearing mice were injected with 1 × 109 N+R@PLTs and then randomly divided into two groups: One group was irradiated at the tumor site with NIR laser (0.65 W/cm2, 5 min) at 1 hour after N+R@PLT injection, while the other group was not. Twelve hours later, the tumors of both groups were collected and frozen in optimum cutting temperature (OCT) tissue compound (Sakura, Tokyo, Japan) on dry ice and then sectioned into 10-μm slices. The obtained tumor slices were incubated with CD31 and CD62P antibodies, followed by orderly staining with fluorescence secondary antibody and 4′,6-diamidino-2-phenylindole (DAPI) solution (0.1 μg/ml). Last, the slices were imaged with an automatic multispectral imaging system (PerkinElmer).

For R, N, and nPLT colocalization, Balb/c mice were treated in the same way as mentioned above except that the tumor-bearing mice were injected with N+R@PLTs in which R and N were labeled as mentioned in SIM experiment. These mice were randomly divided into two groups after injection: One group was irradiated with 808-nm NIR laser at the tumor site at 1 hour after injection, while the other group was not. Twelve hours later, the tumors of both groups were collected and frozen in OCT tissue compound on dry ice and then sectioned into 10-μm slices. The obtained tumor slices were incubated with CD62P antibody, followed by orderly staining with fluorescence secondary antibody and DAPI solution (0.1 μg/ml). Last, the slices were imaged with an automatic multispectral imaging system (PerkinElmer).

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