Fitc labeled anti mouse cd4

To evaluate the therapeutic effect of S1P/JS‐K/TMZ/Lipo, the mice were randomly divided into four groups as follows: PBS, JS‐K/Lipo, TMZ/Lipo and S1P/JS‐K/TMZ/Lipo. All the mice were intravenously injected with PBS (100 µL), JS‐K/Lipo (100 µL), TMZ/Lipo (100 µL) and S1P/JS‐K/TMZ/Lipo (100 µL). Tail vein injections treatments were performed every 2 days, for a total of five injections. Body weight was recorded every other day for 16 days. Finally, mice were sacrificed to collect tumors with major organs (heart, liver, spleen, lung, and kidney) to analyze T cell infiltration and tumor apoptosis with different treatments. First of all, the tissue sections of 3 µm thickness from tumors were soaked in acetone for 15 min and then gently washed with PBS. Then, the sections were blocked with 5% BSA and incubated with antibodies against FITC labeled anti‐mouse CD11c (1:250, Biolegend), and FITC labeled anti‐mouse Foxp3 (1:250, Biolegend), FITC labeled anti‐mouse CD4 (1:250, Biolegend), FITC labeled anti‐mouse CD8 (1:250, Biolegend) for 4 h. Next, sections were gently washed three times by PBS and stained with DAPI. Finally, CLSM was applied to collect images.