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.