Tcs sp8 confocal laser microscopy

4T1 cells and immune cells extracted from the spleen of mice were inoculated into confocal dishes overnight, afterward the culture medium was replaced with fresh ones containing (I) PBS, (II) BSA-Man-Ft, (III) BSA-Man-Ft@Lap, (IV) BSA-Man@Mn²⁺-Ft, and (V) BSA-Man@Mn²⁺-Ft@Lap at the equivalent concentration of 100 μg/mL, while the pH value of medium was adjusted to 6.5. After incubation for 24 h, cells attached on the dishes were washed with PBS for 3 times and incubated with FDA (50 μg/mL) for 30 min. Then the cells were again washed with PBS for 3 times and treated with propidium iodide (400 μg/mL) in dark for 10 min. The cells were washed with PBS for another 3 times and observed with Leica TCS SP8 confocal laser microscopy at ×40.

After indicated treatments with mice in the figure legends, spleen and lymph nodes were fixed overnight at 4°C in PBS-buffered 1% paraformaldehyde, embedded in OCT compound (Tissue-Tek), and kept at -80°C until further processing. Eight-micrometer cross-sections through the tissue midline were prepared and permeabilized with 0.3% TritonX-100 in PBS for 10 min and blocked with 5% BSA in PBS for 30 min at room temperature. Sections were incubated with antibodies against the following proteins: Phospho-Histone H2A.X(Ser-139) from cell signaling technology (#2577); Anti-Mouse CD4 APC (eBioscience #17-0042-82); Hoechst 33342 (Beyotime #C1029); and Goat anti-Rabbit IgG(H+L)-AF488 (Thermofisher Scientific #A11034). Imaging of the sections was carried out using Leica TCS SP8 confocal laser microscopy under a ×63 oil objective.