2.3. Immunohistochemical staining
7 days after MPTP injection, the animals were anesthetized with isoflurane, perfused with heparinized PBS solution through the heart, and then perfused with 4% paraformaldehyde and PBS solution. Then the whole brain was fixed overnight in 4% paraformaldehyde, and then in 20% and 30% sucrose solution prepared by PBS for 48 hours respectively. After that, the brain was frozen on ice and continuous coronal frozen sections were made with a thickness of 30 µm. The obtained brain tissue sections were placed in 50% glycerol/PBS solution and stored at -20 °C. For fluorescence staining, sections from the same position of SN of mice in each group were washed in PBS, blocked in 5% BSA, and then used with anti-F4/80 (cat#157304, Biolegend), anti-CD45 (cat#157214, Biolegend), anti-CD11c (cat#157305, Biolegend) or anti-CD31 (ab281583, Abcam) antibodies. Shake gently overnight at 4 °C. The next day, it was washed three times in PBS for 5min, and then applied to the appropriate secondary fluorescent antibody for 1 hour at room temperature. Then, the slices were installed on the coated glass slide and sealed with anti-fluorescence quencher.
For immunofluorescence of mouse and human T cells, monocytes and macrophages, after the purity of the cells sorted by magnetic beads was identified, they were fixed in a centrifuge tube by 4% paraformaldehyde for 10 minutes, and washed with PBS after centrifugation. CXCR4, 6×His, HMGB1, CXCL12 antibodies were added and stained overnight at 4°C. After 3-time washes with PBS, the fluorescent secondary antibodies were added for 1 h at room temperature in the dark, and washed 3 times with PBS. Resuspend in an appropriate volume of anti-fluorescence quencher. Finally, photographs were taken using a super-resolution fluorescence microscope.