Immunofluorescence confocal imaging

To further delineate the trajectories of FOLactis in vivo, we harvested tumors, TDLNs, and main organs, including the heart, liver, spleen, lung, and kidney, after i.t. injection of DiO-labeled FOLactis at the required time. After washing with PBS, the frozen sections were mounted with DAPI (Beyotime, Shanghai, China) and then imaged on a confocal laser scanning microscopy (Leica, Germany). To evaluate the internalization of FOLactis into DCs, DiO-labeled FOLactis (1 × 10⁹ CFU per mouse) were intratumourally injected and tumors were excised 24 h later. We prepared tumor slices, blocked these tumor sections with 2% BSA for 30 min, and stained them with anti-CD11c-PE antibody and anti-CD103-APC antibody overnight. Finally, the tumor slices were imaged using a confocal laser scanning microscope (Leica, Germany). The fluorescence intensity of CD11c⁺ CD103⁺ DCs was statistically analyzed using Image-Pro Plus 6.0.