Journal: Scientific Reports

Article Title: fMRI indicates cortical activation through TRPV1 modulation during acute gouty attacks

doi: 10.1038/s41598-019-48656-6

Figure Lengend Snippet: Cortical TRPV1 expression was enhanced by gouty arthritis. ( A ) ROI selection. ( B ) Cortical TRPV1 expression on the MSU-affected and control sides. ( C ) TRPV1 immunoreactivity intensity. ( D ) TRPV1 immunoreactivity coverage. ( E ) Immunoprecipitation of beta-tubulin with TRPV1 immunoblotting, which demonstrates a strong interaction between TRPV1 and neuronal filament proteins. The cropped images of the gels are used in the figure, and images of the full-length gels are presented in Supplementary Fig. S2. (***Indicates statistically significant difference with p < 0.001 after ANOVA). Scale bar = 50 µm.

Article Snippet: The primary antibodies used were: anti-TRPV1 (1:2000; PC420; Merck Millipore, Darmstadt, Germany); anti-COX-2 (1:1000; Thermo Fisher Scientific); β-tubulin (1:2000; MCA2703; Bio-Rad Laboratories); β-actin (1:4000; sc-47778; Santa Cruz Biotechnology).

Techniques: Expressing, Selection, Control, Immunoprecipitation, Western Blot

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: Ventral midbrain NSPC isolated from E12 rat embryos are multipotent. (A) VM NSPC cultured in the presence of the mitogenic factor FGF-2 express the markers of undifferentiated cells: Sox2, Vimentin and Nestin in proliferation stage. (B) After 6 days without FGF-2 (differentiation stage), VM NSPC differentiate into the three lineages of Central Nervous System: neurons (β-III Tubulin + and MAP2+), astrocytes (GFAP+) and oligodendrocytes (O4+), confirming the multipotency of these cultures. Nuclei were stained with Hoechst. Scale bars = 100 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Isolation, Cell Culture, Staining

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA promotes neuronal differentiation but decreases dopaminergic neurons in cultured VM NSPC. After proliferation, cells were kept on differentiating conditions for 6 days (N2 medium without FGF-2) and treated daily with different concentrations of HA (from 1 μM to 1 mM HA). VM NSPCs were analyzed after differentiation. (A) Micrographs showing the neuronal marker β-III Tubulin in control and 10 μM HA-treated cultures. Nuclei were stained with Hoechst. (B) Quantification of β-III Tubulin labeled cells relative to the total number of cells, showing a significant increase in the proportion of β-III Tubulin + cells caused by treatment with 10 μM HA. (C) Micrographs of double immunocytochemistry to detect the neuronal marker β-tubulin III and the dopaminergic marker Tyrosine Hydroxylase (TH) in control and 1 mM HA-treated cultures. Nuclei were stained with Hoechst. Scale bar = 100 μm. (D) Graph showing the percentage of TH-positive neurons in control and after HA treatments, relative to the total number of β-Tubulin III-positive cells. TH + neurons were significantly decreased after treatment with 1 mM HA. **p < 0.01. (E) Micrographs showing the dopaminergic marker TH in control cells, and the decrease caused by 1 mM HA. The H1R antagonist chlorpheniramine was tested either with or without HA. Nuclei were stained with Hoechst. (F) HA-induced decrease of TH-positive numbers was antagonized by chlorpheniramine. Note that the percentage of TH-positive cells is lower than in Figure D because it is normalized by the total number of cells. *p < 0.05 relative to control; ## p < 0.01 compared to 1 mM HA. Scale bars = 100 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Cell Culture, Marker, Control, Staining, Labeling, Immunocytochemistry

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA injection decreases dopaminergic neurons without affecting β-III Tubulin. (A) Coronal sections of the ventral midbrain from E14 vehicle- and HA-injected rat embryos stained to detect neurons (β-III Tubulin+) and dopaminergic phenotype (TH+); a representative section of a vehicle-injected embryo showing many TH-positive neurons while in a similar midbrain section from a representative HA-injected embryo less TH-positive cells are present. Nuclei were detected with Hoechst. Inner white squares in the merge images represent the higher magnifications shown at the bottom. Scale bars: 150 μm and 50 μm for the low-power and high-power pictures, respectively. (B) β-III Tubulin and TH expression by qRT-PCR from E14 vehicle- and HA-injected embryos, with β-III Tubulin presenting no change, and a significant decrease in TH caused by HA. **p < 0.01. (C) Densitometric analyses of the effect of HA administration on protein levels of β-III Tubulin and diverse factors involved in dopaminergic specification and phenotype. HA injection decreased the protein level of TH, Lmx1a, Lmx1b and Pitx3 in midbrain tissue from E14 HA-injected embryos without affecting the generation of neurons, compared to the vehicle-injected condition. Values were normalized to GAPDH signal. *p < 0.05.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining, Expressing, Quantitative RT-PCR

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA does not alter GABAergic or serotoninergic neurons. (A) Coronal sections of the ventral midbrain from E14 vehicle- and HA-injected rat embryos stained to identify neurons (β-III Tubulin+) and GABA-synthesizing cells (anti-GAD65/67+ antibody); vehicle-injected embryos had the same proportion of GAD65/67+ cells when compared to HA-injected embryos. Nuclei were stained with Hoechst. Scale bar: 150 μm. (B) HA did not modify the level of GAD65/67 protein in E14 midbrain tissue compared to vehicle-injected embryos by Western blot. The graph represents the densitometric analysis of GAD65/67 protein content where no significant effects were found; values were normalized to GAPDH. (C) Coronal sections of the ventral midbrain from vehicle- and HA-injected E14 embryos stained with β-III Tubulin and serotonin antibodies, where no differences in serotoninergic neurons are observed. Scale bar: 150 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining, Western Blot

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA acts on early dopamine neural precursors but differentiated dopamine neurons are resistant. Sagittal sections of the VM from vehicle- and HA-treated rat embryos that were injected at different developmental stages, and analyzed two days later. Sections were stained to detect neurons (β-III Tubulin+) and dopaminergic phenotype (TH+); nuclei were detected with Hoechst. The decreasing effect of HA injection on dopaminergic phenotype is evident only when injected at early developmental stages (E10-E12 or E12-E14) but not at later developmental stages (E14-E16 or E16-E18). For the vehicle-injected E10-E12 embryos, only a few TH + neurons were found in the isthmic region and these neurons were absent in HA-injected organisms. Scale bars are indicated in the figure.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: High doses of chlorpheniramine affect normal embryonic development but neither chlorpheniramine nor cimetidine affects dopaminergic neurons. (A) Micrographs from E14 rat embryos injected at E12 with different doses of the H 1 R antagonist, chlorpheniramine. Fifty and twenty-five micrograms interfered with normal development, while 15 μg allows normal development of the embryo. (B) TH and β-III Tubulin staining in coronal sections of the VM from E14 rat embryos injected with 15 μg of the H 1 R antagonist, chlorpheniramine or 50 μg of the H 2 R antagonist, cimetidine. The staining patterns were not modified by these antagonists. Scale bar: 150 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining, Modification

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: The H 1 R antagonist chlorpheniramine, but not the H 2 R antagonist cimetidine, abolished the deleterious effect of HA administration on dopaminergic differentiation. (A-D) TH and β-III Tubulin staining in coronal sections of the VM from E14 rat embryos with the indicated treatments. (A) Normal pattern of dopaminergic neuron staining in a vehicle-injected embryo. (B) Decrease in TH immunoreactivity due to HA administration. (C) Protective effect of the H 1 R antagonist chlorpheniramine on HA-induced decrease of TH immunoreactivity. (D) Administration of HA and the H 2 R antagonist cimetidine did not modify dopaminergic neurons compared to HA-injected embryos. Scale bar: 150 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Staining, Injection

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: Primer sequences for detection of transcripts by qRT-PCR

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques:

Journal: Scientific Reports

Article Title: The Role of Transthyretin in Oligodendrocyte Development

doi: 10.1038/s41598-020-60699-8

Figure Lengend Snippet: TTR influences OPC differentiation. Representative images of neurospheres in 3D culture, isolated from SVZ derived NSCs of P21 ( A ) wild type and ( B ) TTR null mice. Scale bar 5 mm. ( C ) Quantitation of the potency of the NSC colonies isolated from the SVZ of P21 mice by neural colony-forming cell assays. There was a significant increase in the number of colonies with an average diameter ≥ 2 mm that were isolated from the SVZ of P21 TTR null mice when compared with wild type at the same age. Increases corresponded with a significant decrease in the number of colonies with an average diameter < 2 mm that were isolated from TTR null mouse SVZ compared to wild type. The colonies ≥ 2 mm in diameter are “NSC derived” and have self-renewal and multi-potential capabilities. Colonies < 2 mm diameter are “progenitor derived”. All data were expressed as the mean ± SEM. Statistical comparisons were performed using a one-way ANOVA with post hoc analysis using Sidak’s test as appropriate (data derived from n = 4 independent experiments per genotype and P < 0.05 was considered statistically significant). ( D ) Characterization of NSCs isolated from the SVZ of P21 TTR null mice and differentiated into the three neural lineages: oligodendrocytes, astrocytes and neurons. Nuclei were stained with DAPI; oligodendrocytes were stained with anti-Olig2; neurons were stained with anti-β-III-tubulin; astrocytes were stained with anti-GFAP. Lack of TTR promotes NSCs to differentiate into glial precursor cells. Differentiation assay for NSCs isolated from the SVZ of P21 TTR null mice showed a greater proportion of cells differentiating into a glial lineage, whereas the equivalent cells from wild type mice had a greater proportion differentiating into a neuronal lineage. All data were expressed as the mean ± SEM. Statistical comparisons were performed using a one-way ANOVA with post hoc analysis using Sidak’s test as appropriate (n = 3 independent experiments and P < 0.05 was considered statistically significant). (Modified from ).

Article Snippet: Cell pellets were collected and incubated with primary antibodies diluted in blocking solution: mouse anti-Olig2 (1:200; Merck Millipore, MAPN50), mouse anti-beta III tubulin (1:150; Merck Millipore, MAB1637), mouse anti-GFAP (1:200; Merck Millipore, MAB360), rabbit anti-TTR (1:300;; ABBIOTC, 250892) and incubated overnight at 4 °C.

Techniques: Isolation, Derivative Assay, Quantitation Assay, Staining, Differentiation Assay, Modification