The membranes were then incubated for 2 h with anti-rabbit-HRP IgG for SYN, SYP, BDNF, GluR1 and GluR2/3 and 5-Fluoracil ic50 anti-mouse-HRP

IgG for NFs, MAP2 and GFAP (Amersham, Little Chalfont, Buckinghamshire, UK) diluted 1:10,000 in TTBS with 1% non-fat milk. The probed proteins were developed by using a chemiluminescent kit (ECL, Amersham Biosciences, NJ, USA). The membrane was then incubated for 30 min at room temperature with stripping buffer and an anti-β-actin antibody (Sigma, St. Louis, MO, USA) was used to quantify β-actin as a loading control. The bound antibodies were visualized using radiographic films which were placed in contact with the membranes, then developed and fixed. The quantification of band intensity was performed with Scion Image 4.0.2 (Scion Corporation, Frederick, MD, USA). The hippocampi were collected (8 animals per group) and immediately homogenized in 1 mL TRIzol (Invitrogen, Carlsbad,CA, USA) with a homogenizer and total RNA was isolated following the manufacturer’s suggested protocol. Briefly, following one chloroform extraction step, RNA was precipitated with isopropanol and the pellet washed once in 70% ethanol. After air-drying, RNA was resuspended in DEPC-treated

water and the concentration AZD6244 of each sample was obtained from A260/A280 nm measurements. Residual DNA was removed using DNase I (Invitrogen) by following the manufacturer’s protocol. For each 20 μL reverse transcription reaction, 4 μg total RNA was mixed with 1 μL oligodT primer (0.5 μg/μL; Invitrogen) and incubated for 10 min at 65 °C. After cooling on ice the solution was mixed with 4 μL 5× first strand buffer, 2 μL of 0.1 M DTT, 1 μL of dATP, dTTP, dCTP and dGTP (10 mM each), and 1 μL SuperScript III reverse transcriptase (200 U/μL; Invitrogen) and incubated for 60 min at 50 °C. Reaction was inactivated by heating at 70 °C for 15 min, and the samples were diluted four times. The real-time PCR reaction system included the following: 200 to 400 nM primers, Quinapyramine 5 ng cDNA samples, and 1× SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Using

the Rotor-Gene 3000 Real-time PCR detection system (Corbett Research, Mortlake, NSW, Australia), cycling conditions were set as follows: after initial activation at 50 °C for 2 min and 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min, then melt curve analysis was performed by heating samples from 65 °C to 99 °C (1 °C increment changes at 5 s intervals), in order to evaluate primer specificity. All sample measurements were performed in duplicate. Primers used for the housekeeping genes, hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyltransferase 1 (HPRT1), were described by Depreter et al. (2002) and the primers for the genes of interest were designed using software primer express v3.0 (Applied Biosystems) (Depreter et al., 2002).