Biotinylated goat anti rabbit antibody

Newborn mice were sacrificed at P1 and brain removed, fixed overnight in PBS 4% paraformaldehyde, cryoprotected in 20% sucrose, and rapidly frozen by immersion in isopentane on dry ice. For each brain, a complete set of coronal sections was cut through the SN and VTA at 30 μm. Sections were mounted on slides, blocked, and permeabilized with PBS, 5% BSA, 0.1% Triton for 30 min at RT. They were incubated with an anti-TH rabbit polyclonal antibody diluted in PBS, 1% BSA, overnight at 4°C, followed by a biotinylated goat anti-rabbit antibody (Jackson ImmunoResearch) diluted in PBS, 1% BSA for 30 min at RT, and by the ABComplex and development with DAB. All reagents are detailed in Supplementary Table S4.

Ninety minutes after the last trial, mice were deeply anesthetized with an intraperitoneal injection of ketamine (150 mg/kg) and xylazine (12 mg/kg) and perfused transcardially with saline (0.9%), followed by an ice-cold solution of 4% paraformaldehyde in phosphate buffer (PB 0.1 M, pH 7.4). After postfixation overnight in the same fixative at 4 °C, brains were cryoprotected for 48 hours in a sucrose solution (30% in PB 0.1 M, pH7.4) at 4 °C. 50 µm-thick coronal sections were cut on a freezing microtome and stored in PB 0.1 M solution containing 0.02% of sodium azide.
Free floating sections were rinsed in 0.1 M PB and incubated for 30 min with H₂O₂ hydrogen peroxide (0.3% in PB). After PB rinses, sections were incubated overnight with anti-c-Fos primary rabbit polyclonal antibody (1:1000, Santa Cruz) diluted in blocking solution (PB 0.1 M, 0.1% BSA, goat serum 2%, 0.2% Triton X-100). A biotinylated goat anti-rabbit antibody (1:500, Jackson Immunoresearch) was used as secondary antibody. After washing, staining was revealed using the avidin-biotin peroxidase method (ABC kit, Vectastain Elite kit, Vector Laboratories, Burlingame, CA, USA) coupled to diaminobenzidine as a chromogen. Sections were mounted on gelatin-coated slides.

Peroxidase immunohistochemistry were carried out on cerebellar sections according to our lab protocol, as described previously [18 (link),34 (link)]. Briefly, at two postnatal ages of P5 or P17, animals were transcardially perfused with 10–20 ml of 4% paraformaldehyde in 0.1–M phosphate buffer, pH 7.4. Brains were then dissected free and post–fixed overnight in the same fixative and cryoprotected with 10%, 20%, or 30% sucrose in the optimal cutting temperature (O.C.T.) compound. Free–floating sections were processed for light microscopic level studies, as described previously. All antibodies were diluted in 0.1–M phosphate–buffered saline (PBS, Gibco, Billings, MT, USA) with 10% normal goat serum and 0.3% Triton X–100. Sections were exposed overnight to overnight with primary affinity–purified antibody to one of the dopamine receptors (DRD1, DRD2, DRD3, DRD4, or DRD5 (1:1000)), followed by washing and secondary antibody exposure (goat anti–rabbit IgG or biotinylated goat anti–rabbit antibody (1:200; Jackson, West Grove, PA, United States). The reaction was developed by using either 0.05% diaminobenzidine or 0.01% hydrogen peroxide reaction.