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

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: 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: PLoS ONE

Article Title: Beta-Actin Is Required for Proper Mouse Neural Crest Ontogeny

doi: 10.1371/journal.pone.0085608

Figure Lengend Snippet: (A) Neurofilament expression in Actb +/+ and Actb −/− embryos. Aberrations of neurofilament expression in Actb −/− embryos are visible at the level of all nerves. (B) Beta-III tubulin expression in Actb +/+ and Actb −/− embryos. Aberrations of beta-III tubulin expression in Actb −/− embryos are visible at the level of IX and X nerves and the dorsal root ganglia. (C) Western Blot analysis showing presence of GFP expression (driven by the endogenous promotor) and the lack of beta-actin expression in Actb −/− embryos. V: trigeminal nerve; VII: facial nerve; IX: glossopharyngeal nerve; X: vagus nerve; max.b: maxillary branch; man.b: mandibular branch; op.b: ophthalmic branch; drg: dorsal root ganglia. Panels depicting Actb +/+ and Actb −/− embryos are of the same magnification.

Article Snippet: Antibodies used in this study are mouse anti-neurofilament mAb (clone 2H3) from Developmental Studies Hybridoma Bank; mouse anti-beta-III tubulin mAb (clone5G8) from Promega; anti-digoxigenin-AP Ab from Roche; mouse anti-p75 NGF receptor mAb, rabbit anti-p75 NGF receptor pAb, mouse anti-Histone H3 (phospho S10) mAb, rabbit anti-vimentin mAb, all from Abcam; rat anti-E-cadherin mAb (clone DECMA-1) from Sigma-Aldrich; mouse anti-N-cadherin mAb (clone 3B9), mouse anti-cadherin-11 mAb (clone 5B2H5), anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 488, anti-rabbit Alexa Fluor 594, anti-rat Alexa Fluor 594, anti-mouse Alexa Fluor 647, anti-rabbit Alexa Fluor 647 all from Invitrogen.

Techniques: Expressing, Western Blot

Journal: Regenerative Therapy

Article Title: Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells

doi: 10.1016/j.reth.2014.12.004

Figure Lengend Snippet: Differentiation of three germ layers of xenogeneic-free SEES cell lines A) SEES cells differentiated in vitro via EBs expressed markers of the primary germ layers. Immunohistochemical analyses of markers of the ectoderm (TUJ1), mesoderm (αSMA), and endoderm (AFP) layers are shown. SEES-4: scale bars are 100 μm; SEES-5: scale bars are 200 μm for TUJ1 and 100 μm for αSMA and AFP; SEES-6: scale bars are 100 μm for TUJ1 and αSMA and 200 μm for AFP; SEES-7: scale bars are 100 μm. B) SEES cells differentiated in vivo via teratoma formation. Hematoxylin and eosin staining revealed germ layer derivatives, such as neural tissues, pigmented epithelium (ectoderm), cartilage (mesoderm), and gut epithelial tissues (endoderm). Scale bars are 200 μm.

Article Snippet: To assess the differentiation of the three germ layers, the primary antibodies used for embryoid bodies (EBs) were anti-α-fetoprotein (AFP; 1:200; R&D Systems; MAB1368), mouse (ascites) anti-β-tubulin III (TUJ1; 1:1000; Promega; #G712A), and anti-α-smooth muscle actin (αSMA; 1:400; Sigma; #A2547).

Techniques: In Vitro, Immunohistochemical staining, In Vivo, Staining

Journal: Regenerative Therapy

Article Title: Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells

doi: 10.1016/j.reth.2014.12.004

Figure Lengend Snippet: Characterization of the pluripotency of SEES-2 maintained using a modified conventional hESC culture medium SEES-2 cells were stably maintained over 20 passages on the qualified MEF feeder layer in the modified medium, which contained pharmaceutical-grade recombinant human bFGF (trafermin) and high-dose (35-K) gamma-irradiated KO-SR without antibiotics. A) Typical hESC colony morphology was readily visible. ALP activity was detected. B) SEES-2 cells expressed undifferentiated hESC markers, including OCT4, NANOG, SOX2, SSEA4, and TRA1-60. SEES-2 cells could differentiate into three embryonic germ layers in vitro and in vivo . Scale bars are 200 μm. C) SEES cells that had been differentiated in vitro via EBs expressed markers of the primary germ layers, ectoderm (TUJ1), mesoderm (αSMA), and endoderm (AFP). Scale bars are 100 μm. D) Histological analysis of teratomas containing multidifferentiated tissues derived from SEES-2 cells. Pigmented epithelium (ectoderm), cartilage (mesoderm), and gut epithelial tissues (endoderm). E) Chromosomal analysis of SEES-2 cells cultivated through 16 passages using a modified conventional hESC culture medium showed a normal 46,XX karyotype.

Article Snippet: To assess the differentiation of the three germ layers, the primary antibodies used for embryoid bodies (EBs) were anti-α-fetoprotein (AFP; 1:200; R&D Systems; MAB1368), mouse (ascites) anti-β-tubulin III (TUJ1; 1:1000; Promega; #G712A), and anti-α-smooth muscle actin (αSMA; 1:400; Sigma; #A2547).

Techniques: Modification, Stable Transfection, Recombinant, Irradiation, Activity Assay, In Vitro, In Vivo, Derivative Assay

Journal: PLoS ONE

Article Title: No Dopamine Cell Loss or Changes in Cytoskeleton Function in Transgenic Mice Expressing Physiological Levels of Wild Type or G2019S Mutant LRRK2 and in Human Fibroblasts

doi: 10.1371/journal.pone.0118947

Figure Lengend Snippet: A-B: Neurite parameters of neuronal cultures from LRRK2, GS-LRRK2 (line 2) and their respective non-tg littermate, which were treated with vehicle-control or LRRK2-IN-1 (0.1 M) for seven days (DIV7). Comparison of parameters describing neurite branching (A) included number of branches, number of neurite trees and number of segments. Comparison of neurite length parameters (B) included total neurite length, average neurite length and maximal neurite length. Data represent mean ± SEM and were analyzed with two-way ANOVA. No significant difference was detected. Number of neurons analyzed for cultures obtained from LRRK2 transgenic mice: non-tg = 1339, non-tg + LRRK2-IN-1 = 1609; LRRK2 = 1697, LRRK2 + LRRK2-IN-1 = 1542, n = 4 independent experiments; Number of neurons analyzed for cultures obtained from GS-LRRK2 transgenic mice: non-tg = 1268; non-tg + LRRK2-IN-1 = 1522; GS-LRRK2 = 1526; GS-LRRK2 + LRRK2-IN-1 = 1844, n = 4 independent experiments; C-H: Representative pictures of ß-Tubulin III stained neurons on DIV7 derived from wild type, GS- LRRK2 (line 2), their non-transgenic littermates. Pictures were obtained with the BD Pathway 855 high content Bioimager. C1-H2: Total neurite length (C1-H1) and number of branches (C2-H2) segmentation corresponding to ß-tubulin III staining images (C-H) obtained from Attovision Software.

Article Snippet: At day in vitro (DIV) 3, 7 and 14 neuronal cultures were immunostained with mouse anti-ß-Tubulin, Class III antibody conjugated to Alexa Fluor 488 (1:50; BD Pharmingen) and the nuclear marker Hoechst 33342 (1:2000), both diluted in PBS.

Techniques: Transgenic Assay, Staining, Derivative Assay, Software