Alexafluor 633 goat anti rabbit secondary antibody

The rats were anesthetized with chloral hydrate 2 weeks after PQ injection and perfused with ice-cold 4% paraformaldehyde in PBS. The brain was removed from the rats followed by overnight xation at 4°C in 4% paraformaldehyde in PBS. Fixed brains were cryopreserved in 30% sucrose in PBS for 2 day and frozen in Tissue-Tek Optimal Cutting Temperature embedding medium. Coronal brain slices (30 mm) were prepared at -20°C in a cryostat and picked up on slides followed by adhering at room temperature for 30 min. For TH immunostaining, the slides were incubated in blocking solution (3% BSA, 0.1% Triton X-100 in PBS) for 1 h and rinsed with PBS for 5 min followed by overnight incubating at 4°C with antityrosine hydroxylase antibody (Abcam). The slides were rinsed with PBS for 5 min and incubated in blocking buffer containing Alexa Fluor 633 goat anti-rabbit secondary antibody (ThermoFisher) for 3 h at room temperature. The slides were rinsed with PBS for 5 min six times, mounted with Prolong Gold antifade reagent, and placed at 4°C for 24 h. Alexa Fluor 633 uorescence was measured in the SNpc and the striatum using a confocal laser-scanning microscopic system.