Trkb fc chimera

Recombinant Human IgG isotype control and TrkB Fc Chimera (R&D Systems, Minneapolis, MN) were diluted to 100ng/ul stock solutions in 0.1M phosphate-buffered saline (PBS) and further diluted to 400ng in 20ul of PBS for in vivo use. The TrkB receptor antagonist, ANA-12 was purchased from Sigma Aldrich (St. Louis, MO, USA) and diluted to 120ng/ul in 100% DMSO. This stock was further diluted 3ug per 20ul injection in 1% DMSO/0.6% Tween-20/PBS. Recombinant BDNF was purchased from PeproTech (Cranbury, NJ, USA, catalog Number 450–02) and diluted to 0.33ug/ul in sterile water.

Kynurenic acid was purchased from Sigma. DNQX (Tocris) was kept at a stock concentration of 20 μM in dimethyl sulfoxide (DMSO) (Sigma). Bicuculline methochloride, furosemide, bumetanide, and gabazine were obtained from Tocris. V1R antagonist dGly[Phaa1,d-tyr(et), Lys, Arg]VP was purchased from Bachem. TrkB-Fc chimera was purchased from R&D Systems. Antibodies from commercial sources: KCC2 (1:500), pan-TrkB (1:1,000), and GAPDH (1:5,000) from Millipore; BDNF (Santa Cruz Biotechnology; 1:100 for WB, 1:300 for IHC); Y515 phosphorylated TrkB from Abcam (1:100); and Iba1 from Wako chemicals (1:1,000). NKCC1 antibody (1:500) was generously provided by Dr. R. James Turner (NIH). VP-neurophysin antibody (mouse monoclonal, 1:100) was kindly provided by Dr. Hal Gainer (NIH).

PAECs were treated, under either normoxia or hypoxia, with 1 nM BDNF (R&D Systems), 1μg/ml TrkB-Fc chimera (R&D Systems), 10μM HIF-1α inhibitor, or 1μM 7,8-Dihydroxyflavone (7,8-DHF; Tocris, Minneapolis, MN), a BDNF agonist. Total RNA was isolated from these cells, using the RNeasy micro kit (Qiagen, Valencia, CA). Complementary DNA (cDNA) was synthesized using Transcriptor reverse transcription kit (Roche, Indianapolis, IN), and was amplified using an LC480 LightCycler (ABI; Carlsbad, CA), and primers listed in Table 1. Real-Time PCR was performed in duplicates per cDNA template, and data for all cDNAs in a category (normoxia, or hypoxia; untreated controls or agonist/inhibitor-treated) were pooled for statistical analysis. All PCR reactions went through 60 amplification cycles. The ratio of fold change in expression of the mRNA of interest for each sample was calculated by normalization of cycle threshold [C(t)] values of the target gene (e.g. BDNF, iNOS etc) to the reference gene (S16) using the comparative C(t) (ΔΔC(t)) method. Data are reported as the ΔC(t) and the average ratio of fold change in mRNA of interest corrected for reference gene. Normoxic, untreated controls were used as calibrator for quantification.