Goat anti trkb

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).

Total proteins were extracted from cortical tissues. BCA reagent (Beyotime, Shanghai, China) was used to measure the concentration of extracted proteins. A total of 10 μg protein sample was loaded for polyacrylamide gel electrophoresis (10% gel for TrkB, 12% gel for BDNF), and were transferred to polyvinylidene fluoride membrane (Pall Life Science, Port Washington, NY, USA) in a 200 V electrical field for 30 min. The membrane was first blocked for 1 h using 5% defatted milk powder, and then was incubated with rabbit anti-BDNF (1:2000, Abcam, Cambridge, MA, USA), goat anti-TrkB (1:1000, R&D System, Minneapolis, MN, USA) or rabbit anti-tubulin (1:2000, Abcam) primary antibody at 4°C overnight. Excess antibody was then washed by PBST, followed by incubation in horseradish peroxidase (horseradish peroxidase-conjugated goat anti-rabbit IgG (1:8000, ServiceBio, Wuhan, China) or mouse anti-goat IgG (1:10 000, ServiceBio) for 1 h at room temperature. The membrane was developed in ECL chromogenic substrates, and was imaged by an automatic protein imaging system (Bio-Rad, Hercules, CA, USA). Image J (NIH, Bethesda, MD, USA) software was used to measure optical density values of each band, which was normalized to that of tubulin. Each experiment was performed in triplicates.

BLA and IL brain tissue samples were removed and lysed with TNE buffer. The protein concentration of each sample was detected by the BCA reagent (Thermo). For analysis of TrkB phosphorylation (p-TrkB), 5 mg protein was utilized for immunoprecipitation with the rabbit anti-TrkB antibody (1:200, Millipore), followed by immunoblotting with anti-phospho-tyrosine pY99 (1:3000, Santa Cruz), goat anti-TrkB (1:2000, R&D) and rabbit anti-β-actin (1:1000, Sigma) antibodies. Ratios of p-TrkB/total TrkB derived from control groups were normalized to 1.0.