6 μg/ml), Linsitinib price in 0.1% bovine serum albumin in 0.1 M Tris saline (pH 7.6) for 1 day at room temperature and an additional 3 days at 4°C. The primary antibodies were visualized by the immunoperoxidase method. Sections were analyzed on a Tecnai Biotwin transmission electron microscope (FEI) equipped with an AMT digital camera. Profiles were identified by the morphological
criteria as previously described (Peters et al., 1991). For the quantitative analysis, ten random nonoverlapping micrographs (36 μm2 per micrograph) were taken from the tissue-plastic interface of stratum radiatum of the dorsal hippocampus of each animal (n = 3 per condition). Cultured rat astrocytes and rat hippocampal brain slices were used for western blotting. Cells and brain slices were homogenized using lysis buffer containing the following: 100 mM Tris (pH 7.0), 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 20 mM sodium pyrophosphate, and 0.5% NP40 with phosphatase and protease inhibitor cocktail (Roche). The homogenates
were then centrifuged at 13,000 × g (20 min, 4°C) to remove cellular debris, and then protein concentrations of the crude click here lysates were determined by performing a Bradford assay with the DC Protein Assay dye (Bio-Rad). The protein samples were diluted with 1× Laemmli sample buffer and boiled for 5 min. After SDS/PAGE, proteins were transferred to PVDF membranes, blocked in 5% milk for 1 hr at room temperature, rinsed with Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated GPX6 with mouse anti-sAC monoclonal antibody (R21, 1:2,500) overnight at 4°C. After four washes with TBST, the membranes were incubated with the anti-mouse secondary
antibody conjugated to horseradish peroxidase (1:10,000) for 1 hr at room temperature. The membranes were then washed three to four times (15 min) with TBST, and bands were visualized using enhanced chemiluminescence (ECL, Amersham Bioscience). Total RNAs were extracted from hippocampal brain slices and cultured astrocytes using TRIzol reagent (GIBCO-BRL) and were subjected to DNase I treatment and complementary DNA synthesis was carried out using M-MLV reverse transcriptase (GIBCO-BRL). Reverse transcriptase was omitted as a negative control. PCR primers (Pastor-Soler et al., 2003) are all intron spanning and sequences and expected product sizes are as follows: sAC sense 5`-CATGAGTAAGGAATGGTGGTACTC-3`; antisense 5`-AGGGTTACGTTGCCTGATACAATT-3` (110 bp); β-actin sense 5`-GTGGGGCGCCCCAGGCACCA-3` and antisense 5`-GTCCTTAATGTCACGCACGATTTC-3`(526 bp). Primers used to amplify sAC splice variants were as follows: sAC; i.e., from exons 1 to 5: sense 5`-ATGAGTGCCCGAAGGCAGGAATTACAG-3` antisense 5`-TGCTCTCTGATCCG GAATCCT-3`; sACt from sACfl splice variants; i.e., from exons 9 to 13: sense 5`-TGCAAACCCACTGCTTGCTTGC-3` antisense 5`-ACTCGGCTGCAGTTCGTCA T-3`; sACsomatic, which starts at the alternate promoter upstream from exon 5; i.e.