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Three random fields within the DG were captured per section and three sections per mouse were scored. A total of n = 4 per group: Cx3cr1?/? (VEH or LPS); Cx3cr1?/?/Mapt?/? (VEH or LPS); non-transgenic (KA) were used for the quantification. Values expressed are mean �� SEM number of CC3+ neurons per 500 ��m2. TUNEL positive cells The number of TUNEL positive cells in the granule cell layer of the DG were scored manually per each field (two random fields/section; two sections per genotype) in the max-Z projected confocal digital images taken at 65x magnification. A total of n = 4 mice per group: Cx3cr1?/? (LPS) and Cx3cr1?/?/Mapt?/? (LPS) were used for quantification. The values expressed are the total number of Bcl-2 protein TUNEL positive cells per ��m2, mean �� SEM. Gene expression analysis RNA from the hemi-brain (or from co-cultured Cx3cr1?/? microglia) was extracted using the Trizol Reagent as described by the manufacturer (Life Technologies). Total RNA (100 ng/��L) was converted to cDNA using the High Capacity cDNA Reverse Transcription kit (Life Technologies, 4368813) and amplified using specific TaqMan probes (Life Technologies, 4331182, see Table ?Table1)1) and GAPDH was used as a house-keeping gene for normalization, FRAX597 on the StepOnePlus? Life Technologies Real-Time PCR System. Table 1 List of gene expression assays utilized. Open field test (OFT) The locomotor behavior of mice was recorded with the open field test. Briefly, each mouse was placed in an open field apparatus equipped with 16 photo beams (San Diego Instruments, San Diego, CA), for a single 15-min session. The time spent by each mouse in either the central or peripheral area of the open field was analyzed. Statistical analysis Data are presented as mean �� SEM, unless otherwise noted, the Student's t-test (two-tailed; unpaired) at 95% confidence interval (for two group comparison) or One-Way ANOVA followed by Tukey or Dunnett's post-hoc test (for multiple comparisons) was utilized for statistical analysis. Statistical significance was determined at p Isoxsuprine deficiency of tau in the primary cortical neurons will provide resistance against microglial-mediated neurotoxicity, we performed neuron-microglia co-culture experiments. Briefly, Cx3cr1?/? primary microglia derived from post-natal day 3 pups were cultured until 14DIV (Saura et al., 2003; Bhaskar et al., 2010). After removal of the astrocyte layer, pure primary microglia were seeded onto plates. Concurrently, primary neurons derived from either non-transgenic or tau knockout (Mapt?/?) mouse embryos (E16.5) were cultured until 21DIV. For the co-culture experiment, primary microglia were stimulated with 1 ��g/ml LPS for 24 h. Then, the primary neurons were co-cultured with the LPS-activated microglia for 24 h. Neurons were then fixed and processed for immunofluorescence analysis for A5 and CC3 to determine the extent of neurodegeneration (Figure ?(Figure1A).