Sc 546

NAc and mPFC tissue blocks were harvested with 12-gauge punches and then sonicated in 30 μL homogenization buffer containing 320 mmol/L sucrose, 5 nmol/L HEPES, 1% sodium dodecyl sulfate (v/v), phosphatase inhibitor cocktails I and II (Sigma-Aldrich), and protease inhibitors (Roche Diagnostics). Protein concentrations were determined with a DC protein assay (Bio-Rad). Proteins (20–40 μg protein per lane) were electrophoresed in 10% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were incubated at 4°C overnight with the primary rabbit anti-BDNF (1:1000, sc-546, Santa Cruz) and mouse anti-GAPDH (1:1000, E021010, Earthox) antibodies. The membranes were rinsed and then incubated for 2 h at room temperature with the secondary antibodies conjugated to alkaline phosphatase (1:500; Santa Cruz). The immune complexes were detected with a BCIP/NBT kit (Beyotime). Blots were analyzed with Photoshop software (Adobe Systems, Inc.), and the gray-scale values of protein bands were normalized to those of GAPDH.

Western blotting was performed as described previously [28 (link)]. The protein-transferred membranes were incubated overnight at 4°C with primary antibodies for TrkB (1:200, sc-8316, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-TrkB (1:500, ab197072, Cambridge, MA, USA), BDNF (1:200, sc-546, Santa Cruz Biotechnology), MMP-2 (1:200, sc-10736, Santa Cruz Biotechnology), MMP-9 (1:200, sc-6840, Santa Cruz Biotechnology), E-cadherin (1:200, sc-7870, Santa Cruz Biotechnology), vimentin (1:200, sc-6260, Santa Cruz Biotechnology), SLUG (1:200, sc-15391, Santa Cruz Biotechnology), SNAIL (1:200, sc-10433, Santa Cruz Biotechnology), twist (1:200, sc-15393, Santa Cruz Biotechnology), VEGF-C (1:200, sc-7133, Santa Cruz Biotechnology), VEGF-D (1:200, sc-7603, Santa Cruz Biotechnology), p-MEK-1/2 (1:200, sc-7995, Santa Cruz Biotechnology), Erk1/2 (1:200, No9102 Cell signaling Technology), p-Akt1/2/3 (1:200, sc-101629, Santa Cruz Biotechnology), or Akt1/2/3 (1:200, sc-8312, Santa Cruz Biotechnology). Peroxidase-linked secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA) were subsequently added and the membranes were further incubated for 1 h at room temperature. The antibodies for α-tubulin (1:1000, Sigma-Aldich, St. Louis, MO, USA) and β-actin (1:200, sc-47778, Santa Cruz Biotechnology) were used as protein loading controls.

WT mice were killed through cervical dislocation and their brains removed and frozen by immersion into isopentane cooled to ∼−50 °C on dry ice. Coronal sections were then cut at a thickness of 20 μm on a cryostat at the level corresponding to NAc (NAc; AP: +1.4 to +0.6 mm), thaw mounted on glass slides and stored at −20 °C until used for immunofluorescent staining. Immunofluorescence was carried out on the fresh–frozen, 20-μm thaw-mounted sections using the Tyramide Signal Amplification (TSA) Plus system (Perkin Elmer, NEL704A001KT). In brief, all slides were fixed using ice-cold 4% paraformaldehyde pH 7.4 (10 min), permeablized in 0.01% Triton X-100 in 0.1 M PBS (5 min) and incubated in 2% hydrogen peroxide diluted in 0.1 M PBS (vol/vol) (15 min). Subsequently, all slides were then blocked (1 h) using the TSA blocking buffer and incubated overnight (4 °C) in rabbit antibody to BDNF (1:500, Santa Cruz Biotechnology, sc-546), washed (3 × 5 min in 0.1 M PBS with 0.05% Tween-20, vol/vol) and incubated for 1 h at 21–23 °C with horseradish peroxidase-conjugated goat antibody to rabbit IgG (1:500, 711-036-152, Jackson ImmunoResearch). Slides were again washed and the TSA reaction was performed to detect BDNF as described in the kit (TSA-Alexa Fluor 488 1:50). Slides were coverslipped in ProLong Gold antifade reagent with DAPI (P-36931, Invitrogen).