Immunohistochemistry

Two fixed embryos from three different pregnant rats (three experiments) per group were used (E14 and E16). On the other hand, one female and male were obtained from four different litters, for a total of eight offspring from four independent experiments at P21. The quantitative immunostaining and TH-positive cells analysis was performed at P21 with four consecutive slices taken between Bregma -5.6 to -5.8 mm (Paxinos and Watson, 2013).

Slices from embryos or brains (P21 animals) were washed with PBS containing 0.1% albumin, blocked and permeabilized (10% normal goat serum, NGS, and 0.3% Triton-X100 in PBS), and incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-H₁R (1:250, RRID:AB_2277328, Santa Cruz Biotechnology, Dallas, TX, United States), anti-LMX1B (1:200, RRID:AB_1269316, Abcam, Cambridge, MA, United States), anti-PITX3 (1:200, RRID:AB_2165300, Abcam) and anti-TH (1:200, RRID:AB_297840, Abcam), and mouse monoclonal anti-NES (Nestin; 1:200, RRID:AB_370407, GeneTex, Irvine, CA, United States), anti-TUJ1 (1:2000, RRID:AB_2210524, Merck Millipore, Burlington, MA, United States) and anti-MAP2 (1:500, RRID:AB_369978, GeneTex). After washing with PBS, slices were incubated for 1 h with the following fluorescent secondary antibodies: Alexa Fluor 488 anti-rabbit IgG (1:1000, RRID:AB_143165, Thermo Fisher Scientific, Waltham, MA, United States), Alexa Fluor 568 anti-mouse IgG (1:1000, RRID:AB_2534072, Thermo Fisher Scientific) or IRDye 800CW donkey anti-rabbit (1:1000, RRID:AB_621848, LI-COR Bioscience, Lincoln, NE, United States). Nuclei were stained with DAPI (5 ng/ml, Sigma-Aldrich) or DRAQ5^TM (5 μM, Abcam) as internal controls and preparations were mounted in Aqua PolyMount (Polysciences, Warrington, PA, United States). For negative controls primary antibodies were omitted.

Microphotographs were obtained using an epifluorescence microscope (Olympus IX81) with a charge-coupled device camera (ORCA-Flash 2.8 CCD model {"type":"entrez-nucleotide","attrs":{"text":"C11440","term_id":"1536511"}}C11440 Hamamatsu; Hamamatsu, Honshu, Japan). For the quantitative analysis of TH signals in brain slices (P21 animals), slices were scanned in the Odyssey CLx Imaging system at 800 nm and the fluorescence was evaluated using the Image Studio 4.0 software (LI-COR Bioscience). Images were processed with Adobe Photoshop CS6 (San Jose, CA, United States).