TH analysis
Rats were sacrificed by an overdose of isoflurane. Striata were solubilized and homogenized in lysis buffer (RIPA buffer, protease and phosphatase inhibitor cocktail) and centrifuged at 18,000 x g for 15 min at 4°C (Pisanò et al. , 2020). Supernatants were collected and total protein levels were quantified using the bicinchoninic acid protein assay kit (Thermo Scientific). Thirty micrograms of protein per sample were separated by SDS-PAGE, transferred onto polyvinyldifluoride membrane and incubated overnight (4°C) with the rabbit anti-TH primary antibody (Merck Millipore, AB152, 1:1000). Membranes were then washed and incubated with horseradish peroxidase-linked secondary antibody (Merck Millipore, goat anti-rabbit IgG HRP-conjugate 12-348, 1:4000). Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL) detection kit (Pierce™ BCA Protein Assay Kit, Thermo Scientific or ECL+, GE Healthcare). Images were acquired and quantified using the ChemiDoc MP System and the ImageLab Software (Bio-Rad). Membranes were then stripped and re-probed with rabbit anti-α-tubulin antibody (Merck-Millipore 04-1117, 1:25000). Data were analyzed by densitometry and the optical density of TH protein band was normalized to α-tubulin levels.