Goat anti rabbit igg horseradish peroxidase conjugate antibody

Mice were decapitated following cervical dislocation, the brain was quickly removed, placed in ice cold PBS (phosphate-buffered saline) and then positioned on a brain mold, where 1.5 mm slices were taken containing the mPFC and BC. The slices were placed on dry ice, and the prelimbic area of mPFC was dissected out and stored at -80 o C. The BC was also isolated from the corresponding slices and stored at -80 o C. Frozen tissue blocks were lysed in a solution containing (in mM) HEPES 50, NaCl 150, MgCl2 1.5, EGTA 5, Glycerol 1%, Triton-X100 1%, 1:1000 protease inhibitors cocktail. Proteins ran on 8.5% bis-acrylamide gel and were transferred onto a nitrocellulose membrane (Whatman GmbH, Dassel, Germany). The membrane was blocked, incubated in rabbit polyclonal anti-K+/Cl-Cotransporter (KCC2) (Merck KGaA, Darmstadt, Germany, 1:1000) or rabbit monoclonal anti-GAPDH (Cell Signaling Technology Europe BV, Leiden, Netherlands, 1:1000), washed, incubated in secondary goat anti-rabbit IgG Horseradish Peroxidase Conjugate antibody (Invitrogen, 1:5000), and digitally exposed using the Molecular Imaging system ChemiDoc (BioRad Laboratories, Inc, California, U.S.A.). Analysis of KCC2 and GAPDH expression was performed with ImageJ software, and the raw values of KCC2 from each sample were normalized to their respective GAPDH values.

Mice were decapitated following cervical dislocation, and the brain was quickly removed, placed in ice-cold phosphate-buffered saline (PBS), and then positioned on a brain mold, where 1.5-mm slices were taken containing the PFC and BC. The slices were placed on dry ice, and the prelimbic area of PFC was dissected out and stored at Ϫ80°C. The BC was also isolated from the corresponding slices and stored at Ϫ80°C. Frozen tissue blocks were lysed in a solution containing, in mM, 50 HEPES, 150 NaCl, 1.5 MgCl 2 , 5 EGTA, 1% glycerol, 1% Triton X-100, 1:1,000 protease inhibitor cocktail. Proteins were run on 8.5% bis-acrylamide gel and transferred onto nitrocellulose membrane. The membrane was blocked, incubated in rabbit polyclonal anti-NR2A (1:2,000; Alomone, Jerusalem, Israel), rabbit polyclonal anti-NR2B (1:2,000; Alomone), or rabbit monoclonal anti-GAPDH (1:1,000; Cell Signaling Technology Europe, Leiden, Netherlands), washed, incubated in secondary goat anti-rabbit IgG horseradish peroxidase conjugate antibody (1:5,000; Invitrogen), and digitally exposed using the molecular imaging system ChemiDoc (Bio-Rad). Analysis of NR2A, NR2B, and GAPDH expression was performed with ImageJ software, and the raw values of NR2A and NR2B from each sample were normalized to their respective GAPDH values.