Mouse anti foxp3

For immunofluorescence staining, the following primary antibodies were used: rabbit anti‐calcium binding adaptor molecule 1 (IBA1) (Wako), goat anti‐GFAP and anti‐NeuN (Abcam), mouse anti‐IL‐6, rabbit anti‐TNF‐α (Santa Cruz), rat anti‐KI67 (Abcam), mouse anti‐FOXP3 (BD), mouse anti‐CD16, and anti‐CD206 (Boster). The slices were imaged using a confocal microscope (Olympus FV1000). Three‐dimensional reconstruction and co‐localization analysis were conducted using Imaris software. Morphological changes in microglia were analyzed with the Sholl analysis plugin for ImageJ. Detailed information about three‐dimensional reconstruction and measurement of the co‐localization coefficient using Imaris software, and the Sholl analysis is thoroughly demonstrated in Appendix S1.

The cells from spleen were harvested from WT and Npr1^−/− mice and washed with cold PBS buffer. Then, the cells were made a suspension of 1 × 10⁶ cells per milliliter in PBS and divided into two groups. One group of cells were incubated with mouse anti-CD45, anti-CD3, anti-CD8, anti-CD11B, anti-Ly-6G, and anti-NK1.1 (BD Biosciences, USA) at 4 °C for 15 min. The other group of cells were first incubated with mouse anti-CD3, anti-CD4, and anti-CD8 at 4 °C for 15 min and then blocked with 1 × TF Fix/Perm working solution for 40 min. Subsequently, the cells were incubated with mouse anti-FOXP3 (BD Biosciences, USA). Finally, the cells from both groups were washed with PBS and inspected using FACSVerse flow cytometer (BD Biosciences, USA). The scatter plots were created and analyzed by FlowJo v10.0.