biotin conjugated polyclonal detection antibody  (R&D Systems)

Journal: The Journal of Neuroscience

Article Title: Cross Talk Between Brain Innate Immunity and Serotonin Signaling Underlies Depressive-Like Behavior Induced by Alzheimer's Amyloid-β Oligomers in Mice

doi: 10.1523/JNEUROSCI.1269-16.2016

Figure Lengend Snippet: Primer sequences used in PCR amplification

Article Snippet: After 24 h, plates were washed extensively and incubated with anti-TNF-α polyclonal detection antibody conjugated with biotin (Peprotech) for 2 h at 25°C followed by streptavidin-HRP (Zymed) for 1 h at 25°C.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Cross Talk Between Brain Innate Immunity and Serotonin Signaling Underlies Depressive-Like Behavior Induced by Alzheimer's Amyloid-β Oligomers in Mice

doi: 10.1523/JNEUROSCI.1269-16.2016

Figure Lengend Snippet: Microglial activation and TNF-α signaling underlie AβO-induced depressive-like behavior in mice. A, B, WT or APP/PS1 mice (8–9 months old) were tested in the FST (A) or tail suspension test (B). n = 7–11 mice per group. C, Swiss mice (3 months old) received a single intracerebroventricular infusion of 10 pmol of AβOs (or vehicle). qRT-PCR was performed 24 h after AβO infusion. n = 8 mice per group. D, E, Brain TNF-α (D) and sTNFR1 (E) levels in hippocampal extracts from AβO-infused Swiss mice. n = 8–10 mice per group. F, Plasma levels of sTNFR1 in AD patients or age- and sex-matched controls. G, H, Mice were injected intracerebroventricularly with infliximab 30 min before AβO infusion. FST (G) or sucrose preference test (H) was performed 24 h after AβO infusion. n = 14–15 mice per group in the FST group; n = 6–7 per group in the sucrose preference test group. I, Representative immunohistochemistry images for Iba-1 (red) in hippocampal sections from AβO- or vehicle-injected Swiss mice. Nuclei are in blue (DAPI). n = 4 per group (20 images per animal). Scale bar, 50 μm. J, Quantification of Iba-1+ cells in the dentate gyrus of mice infused with AβOs or vehicle. K–M, Swiss mice received daily intraperitoneal injections of vehicle (saline) or minocycline (50 mg/kg/d) for 3 d before intracerebroventricular infusion of AβOs or vehicle. qRT-PCR for determination of TNF-α expression was performed 24 h after AβO injection (K). n = 6–8 per group. FST (L) or sucrose preference test (M) was performed 24 h after AβO infusion. n = 17–23 per group in the FST group; n = 6–7 in the sucrose preference test group. N–P, Clodronate- or PBS-containing liposomes were administered intracerebroventricularly to mice in a single dose 3 d before AβO or vehicle infusion. N, Brains were processed for immunohistochemistry 24 h after AβO injection. Hippocampal sections were labeled with anti-Iba-1 (red) and DAPI (blue). Scale bars, 50 μm. n = 3–5 mice per experimental group (20 hippocampal images per animal). O, Quantification of Iba-1+ cells in the dentate gyrus of mice infused with AβOs or vehicle that had been treated previously or not with clodronate. P, FST performed 24 h after AβO injection. n = 9–11 per group. In all panels, bars represent means ± SEM, *p ≤ 0.05, **p ≤ 0.01 (A, B, Welch's t test; C–E, J, Student's t test; F, Mann–Whitney U test; G, K, L, M, O, P, two-way ANOVA followed by Dunnett's multiple-comparisons test).

Article Snippet: After 24 h, plates were washed extensively and incubated with anti-TNF-α polyclonal detection antibody conjugated with biotin (Peprotech) for 2 h at 25°C followed by streptavidin-HRP (Zymed) for 1 h at 25°C.

Techniques: Activation Assay, Quantitative RT-PCR, Injection, Immunohistochemistry, Expressing, Labeling, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: Cross Talk Between Brain Innate Immunity and Serotonin Signaling Underlies Depressive-Like Behavior Induced by Alzheimer's Amyloid-β Oligomers in Mice

doi: 10.1523/JNEUROSCI.1269-16.2016

Figure Lengend Snippet: 5-HT blocks AβO-induced microglial activation and elevated TNF-α production and release. A, B, 5-HT levels measured in the hippocampus (A) or prefrontal cortex (B) 10 d after intracerebroventricular infusion of AβOs in mice. n = 8–10 mice per group. C, Brain 5-HT levels after intracerebroventricular infusion in minocycline-treated or saline-treated mice. Minocycline treatment was performed for 3 consecutive days between 7 and 10 d after AβO infusion. n = 8–9 mice per group. D, Mice received an intracerebroventricular injection of 5-HT (100 pmol) 30 min before injection of AβOs (or vehicle). Brain TNF-α was measured 4 h after injection of AβOs. n = 10–11 per group. E, Tryptophan hydroxylase inhibitor (TPHI) was administered intraperitoneally twice a day (250 mg/kg) for 3 d. Brain TNF-α levels were quantified 24 h after the last injection. n = 6–8 mice per group. Error bars indicate means ± SEM, *p ≤ 0.05, **p ≤ 0.01 (A, B, E, Student's t test; C, D, two-way ANOVA followed by Dunnett's multiple-comparisons test).

Article Snippet: After 24 h, plates were washed extensively and incubated with anti-TNF-α polyclonal detection antibody conjugated with biotin (Peprotech) for 2 h at 25°C followed by streptavidin-HRP (Zymed) for 1 h at 25°C.

Techniques: Activation Assay, Injection

Journal: The Journal of Neuroscience

Article Title: Cross Talk Between Brain Innate Immunity and Serotonin Signaling Underlies Depressive-Like Behavior Induced by Alzheimer's Amyloid-β Oligomers in Mice

doi: 10.1523/JNEUROSCI.1269-16.2016

Figure Lengend Snippet: 5-HT blocks AβO-induced microglial activation and elevated TNF-α production and release. A–D, Purified mouse microglia cultures were pretreated (as indicated) with 1 μm 5-HT for 30 min before the addition of 100 nm AβOs (or vehicle) to the culture medium, followed by incubation for 24 h at 37°C. Representative images of F4/80 immunofluorescence labeling (green) are shown. Scale bar, 50 μm. E, Cell body areas were measured using ImageJ. Error bars indicate means ± SEM; n = 3 independent microglial cultures (3 coverslips per culture; 10 images acquired per coverslip); two-way ANOVA followed by Dunnett's multiple-comparisons test, **p ≤ 0.01. F, Integrated F4/80 immunofluorescence. n = 3–4 independent microglial cultures (3 coverslips per culture; 10 images acquired per coverslip). Error bars indicate means ± SEM; two-way ANOVA followed by Dunnett's multiple-comparisons test, *p ≤ 0.05. G, Microglial cultures were incubated with 100 nm AβOs or vehicle for 2 h before determination of TNF-α mRNA levels. Error bars indicate means ± SEM; n = 3 independent microglial cultures, 2 replicate determinations per experimental condition per culture; *p ≤ 0.05 (Student's t test). H, Microglial cultures were pretreated with 1 μm 5-HT for 30 min and further incubated with 100 nm AβOs (or vehicle) for 24 h before measurement of TNF-α protein levels. Error bars indicate means ± SEM; n = 3 independent microglial cultures, 2–3 replicate determinations per experimental condition per culture; two-way ANOVA followed by Dunnett's multiple-comparisons test, **p ≤ 0.01. I, Cultures were pretreated with 1 μm 5-HT for 30 min and further incubated with 100 nm AβOs (or vehicle) for 24 h to before measurement of nitrite levels. Error bars indicate means ± SEM; n = 3 independent microglial cultures, 2–3 replicate determinations per experimental condition per culture; two-way ANOVA followed by Dunnett's multiple-comparisons test, *p ≤ 0.05.

Article Snippet: After 24 h, plates were washed extensively and incubated with anti-TNF-α polyclonal detection antibody conjugated with biotin (Peprotech) for 2 h at 25°C followed by streptavidin-HRP (Zymed) for 1 h at 25°C.

Techniques: Activation Assay, Purification, Incubation, Immunofluorescence, Labeling