Ix51 12ph

For immunofluorescent assay, the SN of brains were isolated, frozen, and cut into 30 µm slices on a Cryostats (Thermo Fisher, Waltham, MA, USA). Then, the slides were permeabilized and blocked in 2% BSA, incubated with TH antibody (Sigma-Aldrich, USA) overnight, and treated with Cy3-labeled goat anti-chicken IgG for 1 h in the dark. Images were analyzed by using an inverted microscope (IX51-12PH, Olympus) at a magnification of 200×.

For an immunofluorescent assay, the cells were seeded onto chamber slides and fixed with 4% paraformaldehyde for 30 min and the animals were transcardially perfused with 4% paraformaldehyde. Brains were isolated, frozen, and cut into 30 μm slices on a cryostat (Thermo Fisher Scientific, Waltham, MA, USA). Then, the sections and cells were permeabilized with 0.1% Triton X-100 for 30 min and blocked in 2% BSA for 30 min at room temperature. After washing with PBS, the slides and cells were incubated with antibodies against TH (Sigma-Aldrich, USA), DAT (Bioss, China), LC3B (CST, USA), and Parkin (CST, USA) at 4°C overnight and incubated with Cy3-labeled or FITC-labeled goat anti-rabbit IgG (Beyotime, Beijing, China) for 1 h in the dark. Mitochondria and nucleus were labeled with MitoTracker and DAPI, respectively. Images were investigated by using an inverted microscope (IX51-12PH, Olympus) and analyzed using ImageJ software.

A 100-mL sample of the concentrated (2.0 g/mL) Welsh onion root exudate solution was treated three times with ethyl ether, ethyl acetate, chloroform, and n-butanol (ratio of root exudate to organic solvent was 2:1, v/v). The extracts were dried with anhydrous sodium sulfate and a reduced-pressure rotary evaporation apparatus. The residues were redissolved in 5 mL methanol and stored at −20°C [26 ].
A 3 mL sample was taken from each of the four extracts and, after the methanol had evaporated completely, 3 mL sterile water was added for use. As the control, 3 mL methanol was placed in a centrifuge tube and evaporated completely, and then 3 mL sterile water was added to the tube. Each well of a 96-well flat cell-culture plate was filled with 20 μL eggs suspension (about 200 eggs), an aliquot of one of the four onion root extracts (10, 20, 50, 100, 150, or 200 μL), and sterile distilled water to make a final volume of 300 μL. All extract volumes were tested in triplicate. The cell-culture plates were incubated at 26°C in a biochemical incubator, and the hatching of eggs was examined every 2 or 3 days with an inverted biological microscope (Olympus, IX51-12PH, Tokyo, Japan).