Immunohistochemistry
Free-floating immunohistochemistry was conducted using the streptavidin-biotin peroxidase method as previously described (Kelly et al., 2021). In brief, sections were quenched in a solution containing 3% hydrogen peroxide and 10% methanol in distilled water to eliminate endogenous peroxidase activity. Tissue was then blocked to prevent non-specific antibody binding by incubation in a solution containing 3% normal horse serum or normal goat serum (depending on the host species of the secondary antibody) in tris-buffered saline (TBS) with 0.2% Triton X-100 for 1 hour at room temperature. Sections were then allowed to incubate overnight with the primary antibody (Mouse anti-tyrosine hydroxylase, 1:1000, Millipore MAB318; Mouse anti-human-α-synuclein, 1:10,000, Millipore 36-008; Rabbit anti-phospho-α-synuclein (S129), 1:5000, Abcam ab51253; Rabbit anti-α-synuclein-aggregate-specific, 1:8000, Abcam ab209538) which was diluted in 1% serum in TBS with 0.2% Triton X-100. The second day the appropriate biotinylated secondary antibody (Horse anti-mouse, 1:200, Vector BA-2001; Goat anti-rabbit, 1:200, Jackson ImmunoResearch 111-065-144) with 1% serum was allowed to incubate with the sections for 3 hours. Sections were subsequently incubated for a further 2 hours with a streptavidin-biotin-horseradish peroxidase solution (Vector PK-4000). A 0.5% diaminobenzidine tetrahydrochloride (DAB) (Sigma D5637) solution in TBS containing 0.3 µL/mL of hydrogen peroxide was used to develop the tissue staining. Sections were mounted onto gelatin-coated slides, dehydrated in a series of ascending alcohols concentrations, cleared in xylene and coverslipped with DPX mountant.