Gfp 1010

The following commercially available antibodies were used for chromatin immunoprecipitation experiments: Anti-H3K4me3 (Millipore, catalog number 04–745; RRID:AB_1163444), Anti-H3K36me3 (Abcam, catalog number ab9050; RRID:AB_306966), Anti-CREB (Millipore, catalog number 06–519; RRID:AB_310153), Anti-PolII (Ser5-phosphorylated) (Abcam, catalog number ab5131; RRID:AB_449369), Anti-PolII (Ser2-phosphorylated) (Abcam, catalog number ab5095; RRID:AB_304749), Anti-CBP/p300 (Millipore, catalog number 05–257; RRID:AB_11213111), Anti-C/EBPß (Santa Cruz, catalog number sc-150; AB_2260363) or Anti-ATF4 (Santa Cruz, catalog number sc-200; RRID:AB_2058752). The following commercially available antibodies were used for microscopy experiments: anti-RFP (Invitrogen R10367; RRID:AB_2315269), anti-GFP (Aves Labs GFP1010; RRID:AB_2307313), goat anti-rabbit Alexa 555 (Invitrogen A21428; RRID: AB_10561552) and goat anti-rabbit Alexa 488 (Invitrogen A11039; RRID:AB_142924). The following cell lines were used: rat H19-7 (ATCC CRL-2526; RRID: CVCL_H781), mouse Neuro-2a (ATCC CCL-131; RRID:CVCL_0470), human HEK-293FT (Invitrogen R70007; RRID:CVCL_6911), human HEK-293T (ATCC CRL-3216; RRID:CVCL_0063).

To assess the efficiency of the virus injections mice were transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 1x PBS. The brains were dissected and post-fixed in 4% PFA in PBS at 4°C overnight. Free-floating vibratome coronal sections (40 μm) were incubated in a blocking solution of 10% normal donkey serum, 3% bovine serum albumin, 0.2% Triton-X 100, 0.02% sodium azide in 1x PBS for 1–2 hr at room temperature (RT). Sections were then incubated with primary antibodies in the blocking solution overnight at 4°C followed by the appropriate Cy3-congugated (Jackson Labs, ME; 1:1000) and Alexa488 (Invitrogen, OR; 1:1000) secondary antibodies for 2 hr at RT. Twenty minute incubations with Hoechst dye (Invitrogen, OR) at RT were performed to label cell nuclei. GFP-expressing neurons were identified with anti-GFP chicken polyclonal antibody (GFP-1010, Aves Labs) at 1:1000. Images were acquired using high-resolution multi-channel scanning confocal microscopy (LSM 510 Imager Z.1; Zeiss). Confocal 3D scans were carried out with a using Plan-Apochromat 63×/1.4, EC Plan-Neofluoar 40×/1.30 oil immersion, and Plan-Apochromat 20×/0.8 objective lenses at four excitation laser lines. DAPI was imaged with a 405 nm solid state laser and a 445/50 emission filter.

Perfused mouse brain tissues were cut into 30 μm thickness using a Leica cryostat (CM 1950). After incubation in blocking solution (1% horse serum (HS) and 0.3% Triton X-100 in PBS (pH 7.4)), brain sections were incubated with the following primary antibodies: TH (1:1,000; Millipore, MAB318), Iba-1 (1:2000; Abcam, ab178846), Nurr1 (1:1,000), phospho-S129 (1:5,000; ab51253), GFP (1:500; Aves Labs, GFP-1010). For IHC, secondary biotinylated anti-mouse (1:200; Vector Laboratories, BA-2001) or anti-rabbit (1:1,000; BA-1100) IgG antibodies were used followed by series of avidin-biotin complex (ABC; Vector Laboratories) and 3,3’-diaminobenzidine (DAB; Sigma-Aldrich) reactions according to the manufacturer’s instruction. For IF, Alexa Fluor® 568 anti-mouse (1:500; Life Technologies, A11031) and Alexa Fluor® 488 anti-chicken (1:500; Life Technologies, A11039) secondary antibodies were used. Images were acquired using a KEYENCE microscope and quantified as the number or optical density of immunoreactive cells using ImageJ software.

Mice were deeply anesthetized with an overdose of Nembutal (Dainippon Sumitomo Pharma) administered by injection, followed by intracardiac perfusion with 4% paraformaldehyde (PFA) in PBS. Dissected OE and OB samples were further fixed in 4% PFA/PBS. Fixed OE–OB samples were decalcified with 0.5 m EDTA for 7 d, cryoprotected in 30% sucrose in PBS overnight, and then embedded in optimal cutting temperature compound. Cryostat sections (16 μm thick) were collected on glass slides and fixed with 4% PFA/PBS for 15 min. In the anti-Neuropilin 1 (Nrp1) immunostaining, sections were treated with 10 mm citrate buffer, pH 6.0, at 120°C, for 20 min for antigen retrieval. Blocking of sections was performed with 5% donkey normal serum. The primary antibodies used in this study were goat anti-Nrp1 [1:100; catalog #AF566, R&D Systems (RRID:AB_355445)], goat anti-Kirrel2 [1:100; catalog #AF2930, R&D Systems (RRID:AB_2130975)], and chicken anti-GFP [1:500; catalog #GFP-1010, Aves Labs (RRID:AB_2307313)]. Alexa Fluor 488-conjugated donkey anti-chicken IgY [catalog #703-545-155, Jackson ImmunoResearch (RRID:AB_2340375)] and Alexa Fluor 555-conjugated donkey anti-goat IgG [catalog #A-21432, Thermo Fisher (RRID:AB_2535853)] were used as secondary antibodies at 1:200 to 1:250. DAPI (Thermo Fisher) was used at 1:1000.

Chemicals were from Sigma-Aldrich unless otherwise stated. Compound C3 (GW440139A), originally provided by GSK (UK), was re-synthesised at the ARUK Drug Discovery Institute at UCL [28 (link)]. LOXO-292 was purchased from MedChemExpress (USA). Recombinant human GDNF and BDNF were purchased from PeproTech (UK), and GFRα1 was purchased from R&D Systems (USA). RET shRNA constructs were purchased from Genecopoeia in psi-LVRU6GP plasmids with eGreen Fluorescent Protein (GFP) reporter genes and puromycin stable selection markers (MSH025822-LVRU6GP, USA). Antibodies used in immunofluorescence (IF) and western blot (WB) experiments were as follows: RET (C31B4, WB 1:1000, IF 1:250), RET pY905 (3221 1:1000), ERK1/2 (9102 1:1000), pERK1/2 T202/Y204 (9101 1:1000), AKT (9272 1:1000), pAKT S473 (4060 1:1000, Cell Signalling Technology (UK); GAPDH (mab374 1:5000, Merck Millipore, Germany); βIII-tubulin (IF 1:500, WB 1:3000, 302305, Synaptic Systems, Germany), GFP (GFP-1010, IF 1:1000, WB 1:5 000, Aves Labs, USA). The α-p75^NTR used in the kinase inhibitor screen was previously generated and characterised by the authors [8 (link), 29 (link), 30 (link)].