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: International Journal of Molecular Medicine

Article Title: The effect of curcumin on the differentiation, apoptosis and cell cycle of neural stem cells is mediated through inhibiting autophagy by the modulation of Atg7 and p62

doi: 10.3892/ijmm.2018.3847

Figure Lengend Snippet: Growth and fate of newly differentiated NSCs in vitro . (A) NSC neurospheres. (B) Neurospheres or (C) single NSCs in growth medium supplemented with 20 ng/ml EGF and bFGF, expressing Nestin. With 1% fetal bovine serum instead of EGF and bFGF, NSCs differentiated into (D) βIII-tubulin + neurons, (E) GFAP + astrocytes and (F) O4 + oligodendrocytes. Scale bar, 50 µ m. NSC, neural stem cells; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor.

Article Snippet: O7139; Sigma-Aldrich; Merck KGaA), anti-β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.), anti-tubulin βIII (cat. no. MAB1637; Merck KGaA), anti-doublecortin (DCX; cat. no. ab2253) and anti-caspase-3 antibody (cat. no. 9662; Cell Signaling Technology, Inc.).

Techniques: In Vitro, Expressing

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: Comparative morphology and histology of the nasal fossa in four mammals: gray squirrel, bobcat, coyote and white-tailed deer

doi: 10.1002/ar.23352

Figure Lengend Snippet: Epithelial lining of the bobcat nasal fossa. Sagittal view of segmented MRI scans of the bobcat nasal airway showing five coronal sections (b–f) selected for morphological and histological representation (A). The five coronal sections along the rostrocaudal axis illustrate the maxilloturbinal (B–C), nasomaxillary (D) and ethmoturbinal (E–F) regions of the bobcat nasal fossa. Magnified views show the nasoturbinal (G), maxilloturbinal (H, J) and ethmoturbinal (I) covered with nonsensory epithelium and packed with Alcian blue labeled goblet cells at rostral regions of the nasal fossa. Olfactory epithelium covered the nasoturbinal (K), septum (L), and ethmoturbinal (L) in more caudal regions and had the characteristic epithelial thickness and Bowman’s glands in the lamina propria (M) and labeled with β-tubulin III antibody (N–O). nt = nasoturbinal; mt = maxilloturbinal; s = septum; et = ethmoturbinals; ob = olfactory bulb; oe = olfactory epithelium; bg = Bowman’s gland; ne = nonsensory epithelium. Scale bar: B–F = 2 mm; G–L, N = 100 µm; and M, O = 50 µm.

Article Snippet: To further visualize the OE, we conducted immunohistochemistry with a mouse monoclonal anti-neuronal class III β-tubulin antibody (TMJ1, Babco) as has been used to label olfactory neurons in rodents ( Roskams et al., 1998 ), cats (Lishcka et al., 2008) and humans ( Ronnett et al., 2003 ), suggesting reactivity of this antibody in diverse mammalian samples.

Techniques: Labeling

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: Comparative morphology and histology of the nasal fossa in four mammals: gray squirrel, bobcat, coyote and white-tailed deer

doi: 10.1002/ar.23352

Figure Lengend Snippet: Epithelial lining of the coyote nasal fossa. Sagittal view of segmented MRI scans of the coyote nasal airway showing five coronal sections (b–f) selected for morphological and histological representation (A). The five coronal sections along the rostrocaudal axis illustrate the maxilloturbinal (B–C), nasomaxillary (D) and ethmoturbinals (E–F) regions of the coyote nasal fossa. Nonsensory epithelium covered the nasoturbinal (G), maxilloturbinal (H–K), and ethmoturbinal (L) at the rostral regions. Thick olfactory epithelium (L), clearly labeled with β-tubulin antibody (N–O), covered the septum, nasoturbinal, and ethmoturbinal at the caudal regions of the nasal fossa. nt = nasoturbinal; mt = maxilloturbinal; s = septum; et = ethmoturbinal; fs = frontal sinus; ob = olfactory bulb; oe = olfactory epithelium; ne = nonsensory epithelium. Scale bar: B–F = 2 mm; G–L, N = 100 µm; and M, O = 50 µm.

Article Snippet: To further visualize the OE, we conducted immunohistochemistry with a mouse monoclonal anti-neuronal class III β-tubulin antibody (TMJ1, Babco) as has been used to label olfactory neurons in rodents ( Roskams et al., 1998 ), cats (Lishcka et al., 2008) and humans ( Ronnett et al., 2003 ), suggesting reactivity of this antibody in diverse mammalian samples.

Techniques: Labeling

Journal: International Journal of Molecular Sciences

Article Title: Generation of the First Human In Vitro Model for McArdle Disease Based on iPSC Technology

doi: 10.3390/ijms232213964

Figure Lengend Snippet: Primary and secondary antibodies used for the immunocytochemistry analysis of the markers for ectoderm (Tuj1), endoderm (AFP) and mesoderm (SMA) after the in vitro spontaneous differentiation assay.

Article Snippet: Mouse anti-β tubulin isotype III (ectoderm) , 1:300 , Merck, Darmstadt, Germany; #T8660.

Techniques: Immunocytochemistry, In Vitro

Journal: EMBO Molecular Medicine

Article Title: Pre-clinical development of AP4B1 gene replacement therapy for hereditary spastic paraplegia type 47

doi: 10.1038/s44321-024-00148-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse-anti-beta-Tubulin III , Synaptic Systems , Cat# 302304.

Techniques: Control, Variant Assay, Plasmid Preparation, Western Blot, Sequencing, Protease Inhibitor, Lysis, Gel Extraction, SYBR Green Assay, Enzyme-linked Immunospot

Journal: Nanoscale Research Letters

Article Title: A nerve guidance conduit with topographical and biochemical cues: potential application using human neural stem cells

doi: 10.1186/s11671-015-0972-6

Figure Lengend Snippet: Fluorescence microscopy images of hNSCs cultured with PCL conduit for 14 days: a DAPI and b β-III tubulin. DAPI staining is indicated in blue and β-III tubulin staining is indicated in green. Arrow indicates the direction of aligned nanofibers

Article Snippet: After cryosectioning (20-μm slices), each section was washed twice in PBS for 10 min. Then, the samples were treated with blocking buffer (3 % goat serum PBS with 0.01 % Triton X-100) for 1 h at room temperature before adding β-III tubulin goat anti-mouse primary antibody (1:200 dilution, Promega, Madison, WI, USA) overnight at 4 °C.

Techniques: Fluorescence, Microscopy, Cell Culture, Staining

Journal: Nanoscale Research Letters

Article Title: A nerve guidance conduit with topographical and biochemical cues: potential application using human neural stem cells

doi: 10.1186/s11671-015-0972-6

Figure Lengend Snippet: Confocal microscopy images of hNSCs cultured with PSHU-RGD/PCL conduit for 14 days: representative images a , b along the center and e , f inner wall of microchannels, a , d DAPI, b , e β-III tubulin, and c , f combined DAPI and β-III tubulin images. DAPI staining is indicated in blue and β-III tubulin staining is indicated in green . Images show that hNSCs migrated far into the conduit with extensive neurite extension along the nanofiber direction ( arrows )

Article Snippet: After cryosectioning (20-μm slices), each section was washed twice in PBS for 10 min. Then, the samples were treated with blocking buffer (3 % goat serum PBS with 0.01 % Triton X-100) for 1 h at room temperature before adding β-III tubulin goat anti-mouse primary antibody (1:200 dilution, Promega, Madison, WI, USA) overnight at 4 °C.

Techniques: Confocal Microscopy, Cell Culture, Staining