Rabbit anti fabp7

Embryos were fixed in 4% paraformaldehyde for 2 h or overnight at 4 °C, cryopreserved in 20% sucrose, and embedded in OCT compound for cryosectioning. Sections were blocked in 10% Dako serum-free blocking reagent or 10% goat serum in PBS 0.1% TritonX-100 (with some exceptions, see below), followed by incubation in primary antibody for 2 h at room temperature or overnight at 4 °C. Fluorescent Alexafluor-conjugated secondary antibodies were incubated for 1 h at room temperature. Sections were mounted in Prolong Diamond antifade with 4′,6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: Chicken anti-EGFP, 1:1000 (Abcam ab13970); mouse anti-Tuj1, 1:750 (Sigma-Aldrich T5076); goat anti-Sox10, 1:100 (Santa Cruz sc-17342); goat anti-Sox10, 1:100 (R&D Systems AF2864); mouse anti-Isl1, 1:50 (DSHB 40.3A4); rabbit anti-cleaved Caspase-3, 1:500 (Cell Signaling Technologies 9661); rabbit anti-FABP7, 1:200 (Cell Signaling Technologies 13347); goat anti-TrkA, 1:50 (R&D Systems AF1056); goat anti-TrkB, 1:200 (R&D Systems AF1494); goat anti-TrkC, 1:200 (R&D Systems AF1404); mouse anti-AP2α, 1:20, goat serum block (DSHB 3B5); sheep anti-DLL1, 1:200 (R&D Systems AF5026); rabbit anti-p75, 1:250 (Abcam 52987); goat anti-Jag1, 1:200 (R&D Systems AF599); rabbit anti-N1ICD, 1:100 (Cell Signaling Technologies 4147); sheep anti-Notch1, 1:200 (R&D Systems AF5267).

Cell were lysed in RIPA buffer supplemented with cOmplete® Protease Inhibitor Cocktail (Roche) and PhosSTOP® Phosphatase Inhibitor Cocktail (Roche). The lysates were centrifuged at 20,000g at 4 °C for 15 min, and the supernatants were incubated with DTT (100 mM) and NuPAGE® LDS Sample Buffer (Invitrogen) at 70 °C for 10 min. Proteins were separated on Novex® 4–12% Tris-Glycine Mini Gels (Invitrogen) and transferred to a PVDF membrane. The membrane was incubated with 5% skim milk at room temperature for 1 hr and subsequently with primary antibodies at 4 °C for overnight. For the detection of FABP7, the step of centrifuging cell lysates was omitted, and 5% BSA was used as a blocking solution. Primary antibodies were as follows and used at 1:1000 dilution unless otherwise stated: rabbit anti-FABP7 (#13347, Cell Signaling Technology), rabbit anti-UCP1 (U6382, Sigma Aldrich), rabbit anti-PGC-1α (1:200 v/v) (sc-13067, Santa Cruz Biotechnology), rabbit anti-PRDM16 (1:500 v/v) (ab106410, Abcam), rabbit anti-CREB (#9197, Cell Signaling Technology), and rabbit anti-phospho-CREB (#9198, Cell Signaling Technology). Appropriate secondary horseradish peroxidase-linked antibodies were used (Dako, UK). Immunoreactivity was detected with ECL Prime Western Blotting Detection Reagent (Amersham) and visualized using ImageQuant LAS 4000 mini (GE Healthcare).