Under general anesthesia with 1% pentobarbital sodium (40 mg/Kg, i.p.), the mice were intracardially perfused with 30 mL cold PBS (pH 7.4) and 30 mL 4% paraformaldehyde. The brains were removed and post-fixed at 4°C overnight, then kept in 30% sucrose for 2 days. Coronal sections (30 µm) were cut on a freezing microtome (VT1000S, Leica Microsystems). VTA sections were directly imaged for lumafluor, EGFP, and mCherry expression on a laser scanning confocal microscope (FluoView FV1000; Olympus).
For immunofluorescence staining, the sections were blocked for 1 h with 3% donkey serum and 0.25% Triton X-100 in PBS at room temperature. Then they were incubated with the primary antibodies rabbit anti-Kv7.2 (KCNQ2, 1:100; APC-050, Alomone Labs), rabbit anti-Kv7.3 (KCNQ3, 1:100; APC-051, Alomone Labs), and mouse anti-tyrosine hydroxylase (1:600, 318; Millipore), at 4°C overnight. The sections were then incubated for 2 h at room temperature with the secondary antibodies Alexa Fluor 488 anti-rabbit (A-11034), AlexaFluor 594 anti-mouse (A-11032), and AlexaFluor 488 anti-mouse (A-32723) (1:200, Molecular Probes), and rinsed three times with PBS before mounting. Afterwards, the sections were visualized under an Olympus confocal microscope.
For immunofluorescence staining, the sections were blocked for 1 h with 3% donkey serum and 0.25% Triton X-100 in PBS at room temperature. Then they were incubated with the primary antibodies rabbit anti-Kv7.2 (KCNQ2, 1:100; APC-050, Alomone Labs), rabbit anti-Kv7.3 (KCNQ3, 1:100; APC-051, Alomone Labs), and mouse anti-tyrosine hydroxylase (1:600, 318; Millipore), at 4°C overnight. The sections were then incubated for 2 h at room temperature with the secondary antibodies Alexa Fluor 488 anti-rabbit (A-11034), AlexaFluor 594 anti-mouse (A-11032), and AlexaFluor 488 anti-mouse (A-32723) (1:200, Molecular Probes), and rinsed three times with PBS before mounting. Afterwards, the sections were visualized under an Olympus confocal microscope.