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.