Fig. 7

Immunofluorescence staining and flow cytometry assay to analyze tumor-infiltrating lymphocytes in H22 tumor model. Mice were euthanatized when the study ended. The harvested tumor tissues were subjected to immunofluorescence and flow cytometry. a The representative immunofluorescence images and quantitative analysis of tumor-infiltrating CD8⁺ T cells. Harvested tumor tissues were dissociated with Collagenase B and hyaluronidase to prepare single-cell suspensions. Then, the cells were fluorescently stained with the detection antibodies. The representative images and quantitative analysis of tumor-infiltrating b, h CD8⁺ T cells, c, i Ki67⁺CD8⁺ T cells, d, j CD25⁺CD8⁺ T cells, e, k CD69⁺CD8⁺ T cells, f, l Granzyme B⁺CD8⁺ T cells, g, m IFN-γ⁺CD8⁺ T cells. The proportion of tumor-infiltrating lymphocytes in the total live cells was calculated. The size of the scale bar in the immunofluorescence images refer to 100 μm. Bars, SDs; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 denote the significant difference relative to combination treatment. ns: not significant, α-PD-1: anti-PD-1