The vHPC tissues were collected 2 hours after extinction learning (Ext.) or retrieval of fear memory (No Ext.) and were rapidly frozen in liquid nitrogen. Each sample was added to 200 μl of lysis buffer containing 137 mM NaCl, 20 mM tris-HCl (pH 8.0), 1% TERGITOL type NP40, 10% glycerol, complete protease inhibitor set (Sigma-Aldrich, St. Louis, MO), and 0.5 mM sodium vanadate, which was then homogenized and centrifuged at 16,000g for 30 min at 4°C. The supernatants were removed into aseptic Eppendorf tubes and stored at −80°C.

The total protein levels in individual samples were measured by the BCA Protein Assay Kit (Pierce Co., Appleton, WI). The BDNF Emax ImmunoAssay System (Promega Co., Madison, WI) was used to detect the amount of BDNF. Briefly, each well of a 96-well polystyrene plate was incubated overnight at 4°C with 100 μl of anti-BDNF monoclonal antibody (mAb) diluted 1:1000 in carbonate coating buffer [25 mM sodium bicarbonate and 25 mM sodium carbonate (pH 9.7)]. Unabsorbed mAb was removed, and the plates were washed once with tris-buffered saline with Tween 20 (TBST) wash buffer containing 20 mM tris-HCl (pH 7.6), 150 mM NaCl, and 0.05% (v/v) Tween 20. Just before blocking, tissue extracts were removed from the freezer and allowed to cool to room temperature. Plates were blocked using 200 μl of Promega 1× Block and Sample Buffer followed by incubation for 1 hour at room temperature. Plates were then washed using TBST wash buffer. One hundred microliters of each sample or standard (500, 250, 125, 62.5, 31.3, 15.6, 7.8, and 0 pg/ml) was added in duplicates to the plates. The plates were incubated for 2 hours with shaking (600 rpm) at room temperature, which was then followed with five times washing with TBST wash buffer. Anti-human BDNF polyclonal antibody (100 μl of diluted 1:500 in 1× Block and Sample Buffer) was added to each well, and plates were incubated for 2 hours with shaking (600 rpm) at room temperature. Plates were washed again five times using TBST wash buffer. Anti–immunoglobulin G horseradish peroxidase conjugate (100 μl of diluted 1:200 in 1× Block and Sample Buffer) was then added to each well, and plates were incubated for 1 hour with shaking (600 rpm) at room temperature. Plates were emptied again and washed five times using TBST wash buffer. Finally, 100 μl of Promega TMB One Solution was added to the plates. After incubation for 10 min at room temperature, the reaction was stopped using 100 μl of 1 N HCl, which was followed by reading at 450 nm within 30 min. BDNF concentrations were determined on the basis of the absorbance values against the standard curve and reported in pg/mg protein. The values were then normalized to that of the No Ext. group of AAV-Syn-GFP–injected mice in parallel experiments.

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