Proximity Ligation Assay (PLA)

DAT:α-synuclein PLA experiments were carried out using Duolink^® in situ kits supplied by Sigma Aldrich according to the manufacturer’s instructions, and an α-synuclein antibody (syn4D6 ab1903, Abcam), and a DAT antibody (ab5990, Abcam). Briefly, the conjugates were prepared using the DuoLink^® Probemaker kit by incubating 20 μl of each antibody (1 mg/ml) with the Probemaker activated oligonucleotide (Plus and Minus respectively) and conjugation buffer and leaving it overnight at room temperature. Conjugates were incubated with Probemaker stop solution for 30 min at room temperature and then suspended in Probemaker storage buffer. Paraffin-embedded tissue was prepared for fluorescent PLA^® by dewaxing in xylene and histoclear, rehydrating via graded alcohols, blocking endogenous peroxidases with 10% H₂O₂ for 15 min at room temperature, followed by antigen retrieval using microwave heat (10 min total) and citrate buffer pH 6 (ab93678, Abcam). All samples were incubated in Duolink^® block solution for 1 h at 37°C, followed by conjugates diluted in Duolink^® PLA diluent (both DAT and α-synuclein 1:100) overnight at 4°C. After washing in TBS + 0.05% Tween 20, samples were incubated with Duolink^® ligation solutions and ligase for 1 h at 37°C, before washing and incubation with Duolink^® amplification reagents and polymerase for 2.5 h at 37°C. Samples were then washed with Duolink^® wash buffer B and mounted/coverslipped with Fluorsave (Calbiochem). Images were blindly acquired at three distinct sites in dorso-medial, dorso-mid and dorso-lateral striatum from a single 5 μm coronal section per mouse, and puncta were automatically counted with ImageJ. Three mice per genotype were assessed.