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.