Ahb0261

Brain samples were taken from PBS- and AmNa-ASO-treated Line 61 mice and wild type littermates. The hemispheres were divided at the midline. One hemisphere was fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 for 2 hours at RT, cryoprotected in 15% sucrose in PBS overnight, embedded in OCT medium in cryomolds, and snap-frozen in dry ice-cooled liquid isopentane. Frozen samples were stored at −80 °C until sectioning. Frozen brain hemispheres were cryosectioned sagittally at 30 µm thickness on a Leica CM1850. Sections were transferred to 30% sucrose solution in PBS (Thermo Scientific) and stored at 4 °C. For immunostaining, sections were washed and incubated with primary and secondary antibodies as previously described in Furuya et al.^{52 (link)}. In brief, the following primary antibodies were used: phosphorylated α-synuclein antibody, a mouse monoclonal antibody against α-synuclein phosphorylated at Ser129 (Wako, cat # 015-25191, 1:1000 dilution) and Syn211 (Invitrogen, AHB0261, 1:1000 dilution), and a mouse monoclonal antibody that specifically recognizes human α-synuclein. After immunostaining, mounted sections were observed and imaged under a fluorescence microscope (BZ-X700, Keyence).

One series (i.e., every sixth section) was stained with antisera for α-synuclein (α-syn) using the free-floating method, as described previously^{32 (link)}. Tissue was blocked in normal goat serum and incubated overnight in primary antisera directed against wild-type human α- syn (mouse monoclonal anti-human α-syn, Invitrogen AHB0261, 1:2000 dilution for final concentration of 250 ng/ml) in 1.0% normal goat serum (Gibco, Catalog #16210–072). Cell membranes were permeabilized with the addition of Triton-X (0.5%, Sigma X-100) to the 0.1 M Tris buffer during incubations. Sections were then incubated in biotinylated secondary antisera against mouse IgG (Chemicon AP124B, 1:400 dilution for final concentration of 7.5 µg/ml) and followed by the Vector ABC detection kit employing horseradish peroxidase (Vector Laboratories, Burlingame, CA). α-Syn immunoreactive (α-syn-ir) neurons were visualized upon exposure to 0.5 mg/ml 3,3′-diaminobenzidine (DAB) and 0.03% H₂O₂ in tris-buffered saline (TBS). Sections were mounted on subbed slides, dried flat overnight under standard temperature and pressure conditions, dehydrated with ethanol and then xylenes and finally coverslipped with Cytoseal (Richard-Allan Scientific, Waltham, MA).

Guinea pigs were sacrificed by cervical dissection. After removing muscle and anterior segments of the eye, eyecup was fixed using 4% paraformaldehyde (P0099, Beyotime, China) for 0.5 h, followed by impregnating using 10% sucrose in PBS for 2 h, 20% sucrose for 2 h, and then 30% sucrose for 15 h, and finally embedding into Optimal Cutting Temperature (OCT) compound to freeze immediately with liquid nitrogen. The embedded tissue was sectioned into 10 μm thickness for immunofluorescence analysis. Retina slides were blocked with QuickBlock™ solution (P0260, Beyotime, China) for 1 h, then the primary antibodies (Mice anti-SNCA, 1:500, AHB0261, Invitrogen, United States; Rabbit anti TH, 1:1,000, AB152, Sigma-Aldrich, United States) were diluted with a blocking solution (P0262, Beyotime, China) and used to incubate retinas for 16 h at 4°C. After incubating and rinsing, secondary antibodies conjugated to Goat anti-Rabbit-Cy3 (1:1,000, A0516, Beyotime, China) and Goat anti-Mice-Alexa Fluor 488 (1:1,000, ab150113, Abcam, United States) were applied for 2 h at room temperature. Zeiss LSM800 (Carl Zeiss, Germany) and a confocal microscope was used to take micrographs at 10 × 20 fold.