2.10.Western blot
After PBS cardiac perfusion in isoflurane anesthetized mice, midbrain was separated and removed meninges and blood vessels under stereological microscope, and then tissue proteins were extracted by RIPA lysis buffer (P0013B, Beyotime). The protein concentration of the obtained protein was measured by BCA Protein Assay Kit (P0012S, Beyotime). Finally, the protein concentration between samples was adjusted to be uniform according to the BCA results, and the protein Loading buffer was added to the water bath 100℃ for 10min. Then the protein samples were separated in 10% SDS-PAGE and transferred to PVDF membrane. The PVDF membrane were incubated in TBST solution containing TH (#45648, Cell Signaling Technology) and β-Actin (#3700, Cell Signaling Technology) primary antibodies overnight at 4 °C. After washing by TBST for 10min 3 times, PVDF membrane was incubated in HRP-conjugated secondary antibodies (anti-rabbit or anti-mouse 1:10000, Abcam) for 2h at room temperature, after washing by TBST for 10min 3 times, BeyoECL Moon (P0018FS, Beyotime) solution was used for PVDF membrane chemiluminescence detection. Protein banding results were grayscale scanned by Image J.