R 1718 50

Lysates were separated by SDS-PAGE (4%–12% Bis-Tris, Invitrogen, Carlsbad, CA, USA) and transferred to PVDF membrane and probed with antibodies against TrkB (1:1,000, sc-8316, SantaCruz Biotechnology, Santa Cruz, CA, USA) and pTrkB^s478 (1:1,000, R-1718-50, Biosensis), p44/42 MAPK (ERK1/2, 1:1,000, #9102 Cell Signaling Technologies, Danvers, MA, USA) and phosphorylated ERK1/2 (pERK1/2, 1:1,000, #9101 Cell Signaling Technologies, Danvers, MA, USA). All blots shown are representative of at least three independent experiments. Optical density value for each band was determined using FIJI/ImageJ and corrected to loading control and normalized against the relevant control condition.

Doublecortin (DCX) and p-TrkB (S478) immunostaining were performed using a free-floating technique. Randomly selected sections were incubated for 2 d at 4°C with DCX antibody (sc-8066, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:200) and p-TrkB (R-1718–50, Biosensis, Thebarton, Australia, 1:100) in a dilution buffer containing 1 % bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) and 1 % Triton X-100 in 0·1 M phosphate buffer. After washing with phosphate-buffered saline buffer, the sections were reacted with horseradish peroxidase-conjugated anti-goat or rabbit IgG secondary antibody (Vector Laboratories Ltd.) for 2 h at room temperature. After washing the sections again, they were reacted with 0·3 % avidin-biotin–peroxidase complex (Vector ABC kit; Vector Laboratories Ltd.) for 1 h at room temperature. To observe the antigen–antibody reaction, the sections were reacted with 3,3′-diaminobenzidine (Vector Laboratories Ltd.). After dehydration with ethanol at sequential concentrations, the sections were covered with glass coverslips and observed under a Leica DM5500B microscope (Leica Microsystems, GmbH, Wetzlar, Germany).