Alexa fluor 594 red

Two hours after withdrawal from the chronic ethanol diet for 16 days, rats were given intra-NAc infusions of β-endorphin and sacrificed 5 min later. An equal volume of artificial cerebrospinal fluid was administered into the NAc for comparisons of β-endorphin effects. Brains were then prepared for measuring levels of TH immunoreactivity and phosphorylation state in the VTA. Brains were removed after transcardial perfusion of 4% paraformaldehyde solution at the time of euthanasia and allowed to stand in formalin fixative solution for 1 week. Brains were then cryosectioned into 30-μm-thick sections at the level of the VTA (coordinates: anterior, −5.8 to − 6.3 mm; lateral, ±0.8 mm; deep, −8.2 to − 8.5 mm). Coronal brain sections were incubated overnight (approximately 16 hours) at 4°C with rabbit polyclonal antibodies anti-TH (1:500; Millipore, USA) and anti-THser40 (1:500; Millipore, USA), followed by a 2-hour incubation at room temperature with a biotinylated donkey anti-rabbit Alexa Fluor 594 (red; 1:500; Sigma, USA), and mounted onto gelatin-coated slides. The sections were photographed and quantified using confocal laser scanning microscopy (LSM700, Carl Zeiss, Oberkochen, Germany). The three sections were collected at the level of the VTA in each brain for TH immunoreaction. The cells were counted and averaged from three sections.