Af0402

To examine the protein expression levels of the cervical spinal cord, tissues were treated with RIPA lysate (R0020, Solarbio Co., Ltd., Beijing, China) to extract the total protein, and the concentration was determined using the bicinchoninic acid assay as described previously.26 (link) The sample protein (20 μg) was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes, and blocked for 1h with 5% (w/v) bovine serum albumin in Tris-buffered saline at room temperature. Primary antibodies were rabbit α-syn (AF0402, 1:1000, Affinity), synapsin1 (AF6201, 1:800, Affinity), synapsin2 (AF6202, 1:800, Affinity), and mouse β-actin (AF7018, 1:7000, Affinity) and diluted in Tris-buffered saline with Tween 20 buffer overnight at 4°C. The membranes were then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (ZB2301, 1:3000, ZSGB-BIO) for 90 min. Protein signals were visualized using enhanced chemiluminescence (ECL) fluorescence test kit (SC-2048, ZSGB-BIO) at a 1:1 (v/v) ratio. Immunoreactivity was quantified using ImageJ software (National Institutes of Health, Bethesda). The relative protein content was determined by calculating the gray value of the corresponding protein band relative to the β-actin protein band.

The spinal cord tissues were fixed in 4% paraformaldehyde for a minimum of one week and sliced into sections 5-μm thick using a cryostat, then processed for IF. The sections were initially blocked with normal goat serum at 37°C for 30 minutes and subsequently incubated overnight at 4°C with primary antibodies specific to α-syn (AF0402, 1:100, Affinity) and PSD-95 (AF7839, 1:100, Affinity). After rinsing three times with PBS for 5 min each, the sections were incubated with a secondary antibody, and DAPI was used to stain the cell nuclei for 10 min at room temperature. The tissue slices were encapsulated by water-soluble tablets and viewed using a fluorescence microscope (Olympus, Japan). The area of positive staining was measured using Image-Pro Plus analysis software. The mean fluorescence units were calculated by dividing the total fluorescent intensity by the area of each fluorescent particle.