Vectastain anti rabbit

For immunohistochemistry of phospho-Ser129-alpha-synuclein and p62, brains were cut into 30-µm and lumbar (with some cervical and thoracic) spinal cord into 40-µm coronal sections on a vibrating blade microtome (Leica VT1000 S). Free-floating sections were rinsed in phosphate-buffered saline (PBS) at 4°C, followed by 1 h of incubation at 4°C in blocking solution [10% normal goat serum (Vector Laboratories), 0.5% Triton X-100 (AppliChem), PBS] to prevent unspecific staining. Thereafter, sections were incubated with the respective first antibody, either phospho-Ser129-alpha-synuclein (Abcam, dilution 1 : 500) or p62 (Novus Biologicals, dilution 1 : 200) in carrier solution [1% normal goat serum (Vector Laboratories), 0.5% Triton X-100 (AppliChem), PBS] for 48 h at 4°C. Then, sections were washed in PBS and incubated with the appropriate biotinylated secondary antibody for 2 h at room temperature (Vectastain anti-rabbit dilution 1 : 500; Vector Laboratories). Tissue-bound antibodies were visualized by incubating for 90 min with avidin–biotin–peroxidase complex (Vectastain ABC-kit, Vector Laboratories) and Peroxidase substrate Vector NovaRED Substrate Kit (SK-4800 kit; Vector Laboratories). Sections were dehydrated in ascending ethanol concentrations followed by xylene; they were cover-slipped using VectaMount permanent mounting medium (H-5000; Vector Laboratories).