Af7839

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.

Quantitative IHC is a technique used to measure the density of protein expression in immunohistochemically stained images. This method involves calculating the chromogenic density or color and the associated signal intensity to obtain a quantitative result that can be used to study the amount or trend of protein expression in a cell or tissue. The cervical spinal cord of each group was collected and fixed in 4% paraformaldehyde for 3 days at 4°C. Fixed samples were embedded in paraffin, and 5μm-thick sections were obtained. Slices were deparaffinized in xylene and rehydrated in graded alcohol solutions. The tissue sections were repaired in 3% citric acid repair solution under high pressure and then incubated in 3% H₂O₂ for 10 min. Next, the sections were washed with distilled water for 5 min and soaked with phosphate-buffered saline (PBS) for 1min. The sections were then incubated with primary antibody to PSD-95 (AF7839, 1:100, Affinity), GAP-43 (DF7766, 1:90, Affinity) at 4°C overnight. The sections were washed three times with PBS buffer for 5 min on the next day, incubated with the secondary antibody at 37°C for 20 min, followed by reaction termination using 3,3’-diaminobenzidine (DAB). BX43 microscope (Olympus, Japan) was used to capture the immunohistochemistry images of the slices, and Image-Pro Plus software was used to quantify the captured image.

After training and testing, rats were sacrificed and their brains were removed, fixed in 4% paraformaldehyde, and dehydrated in sucrose solution (20%–30%). The tissue was embedded in optimal cutting temperature compound and cut into 10-μm sections. The sections underwent antigen retrieval with citric acid retrieval solution and heating for 10 minutes before being blocked with 1% bovine serum albumin (Sangon Biotech, Shanghai, China). Thereafter, the sections were incubated with primary antibodies against postsynaptic density protein (PSD95; 1:100; Affinity, Changzhou, China; AF7839), microtubule-associated protein 2 (MAP2; 1:200; Affinity; AF4081), and F-actin (1:25; Abcam, Cambridge, UK; ab205) at 4°C overnight, followed by incubation with Cy3- or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA or Abcam) for 60 minutes at room temperature. After being counterstained with 4′,6-diamidino2-phenylindole (DAPI; Aladdin, Shanghai, China), the sections were observed and images of the CA1 region of the hippocampus were captured using a fluorescent microscope (Olympus Optical Co., Tokyo, Japan).