Goat anti unc5d

Embryos were dissected, and the brains were fixed in 4% paraformaldehyde (PFA) for 1–3.5 hr. For postnatal stage, brains were fixed with 4%PFA overnight. Following 30% sucrose replacement, fixed brains were embedded in optimum cutting temperature (OCT) compound, and 20 micrometer slices were cut on a cryostat. The antibodies used were, rat anti-GFP (1∶500; nakalai tesk), rabbit anti-GFP (1∶200; IBL), rabbit anti-DsRed (1∶500; Invitrogen), goat anti-Unc5D (1∶100; R&D), rabbit anti-Tbr2 (1∶300; abcam), goat anti-NeuroD1 (1∶100; Santa Cruz), mouse anti-PCNA (1∶100; Cell Signaling), mouse anti-Tuj1 (1∶500; SIGMA), rabbit anti-Tbr1 (1∶100; abcam), mouse anti-RORb (1∶100; PERSEUS PROTEOMICS), rat anti-Ctip2 (1∶300; abcam), goat anti-Brn2 (1∶100; Santa Cruz), and mouse anti-Prdm8 [24] (link). Alexa Fluor-conjugated secondary antibodies (Invitrogen) were also used. EdU labeling (intraperitoneal injection of 12.5 mg/kg EdU) and staining were performed according to manufacturer's instructions (Invitrogen). Stainings were examined with Zeiss LSM 710 or Olympus IX81, and the images were finally processed with Adobe Photoshop.

Antibodies used in this study include: mouse anti-PLAP (Thermo Fisher Scientific; Waltham, MA), mouse anti-human IgG1-Fc-HRP (Serotec; Raleigh, NC), mouse anti-myc (Abcam; UK, 1:1000), mouse anti-FLAG (Abcam, 1:1000), chicken anti-GFP (Abcam, 1:6000), goat anti-FLRT1 (R&D Systems; Minneapolis, MN, 1:25), rabbit anti-FLRT2 (Abcam, 1:25), goat anti-FLRT3 (R&D Systems, 1:50), goat anti-Unc5A (R&D Systems, 1:25), rabbit anti-Unc5B (Santa Cruz Biotechnology; Santa Cruz, CA, 1:200), rabbit anti-Unc5C (Santa Cruz Biotechnology, 1:50), goat anti-Unc5D (R&D Systems, 1:100), mouse anti-His-HRP (Qiagen; Germany, 1:5000), goat anti-Human IgG (H+L) DyLight 680 (Rockland; Limerick, PA, 1:4000), guinea pig anti-vesicular acetylcholine transporter (VAChT) (EMD Millipore; Hayward, CA, 1:500), mouse anti-neuronal class III beta-tubulin (Tuj1) (Covance; Princeton, NJ, 1:1000), rabbit anti-cocaine- and amphetamine-regulated transcript (CART) (Phoenix Pharmaceuticals; Burlingame, CA, 1:2000), rabbit anti-calbindin (Swant Inc; Switzerland, 1:5000).