Structured Review

Promega anti tubb3 mouse monoclonal antibody
ANA-12 administration reverts EE-induced changes in the meningeal niche. ( A ) Sagittal brain sections of the CD1 mice, showing GLAST + (red) cells above the meningeal basal lamina (green) in the EE VEH and EE ANA-12 mice. ( B ) Sagittal brain sections of the CD1 mice, showing β3-Tubulin + (red) cells above the meningeal basal lamina (green) in the EE VEH and EE ANA-12 mice. ( C ) Graph showing the number of GLAST + cells in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. ( D ) Graph showing the number of β3-Tubulin + cells in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. ( E ) Graph depicting gene expression analysis for Slc1a3 (GLAST) in the NO EE VEH, EE VEH and EE ANA-12 mice. Expression levels are reported as the log 2 fold change with respect to the NO EE VEH mice. ( F ) Graph depicting gene expression analysis for <t>TUBB3</t> (β3-Tubulin) in the NO EE VEH, EE VEH and EE ANA-12 mice. Expression levels are reported as the log 2 fold change with respect to the NO EE VEH mice. ( G ) Representative western blot analysis of mouse brain meningeal protein extracts, which shows the presence of the DCX protein in the EE VEH, EE ANA-12 and NO EE VEH samples (left panel). The graph shows the differential fold change in protein expression for the DCX protein in the NO EE VEH, EE VEH and EE ANA-12 mice with respect to the NO EE VEH mice (right panel). ( H ) Sagittal brain sections of the CD1 mice, showing fractones (*) identified via laminin (green) staining in the brain meninges of EE VEH and EE ANA-12 mice. An insert illustrates laminin staining (fractones) below the meningeal nuclei (TOPRO-3). ( I ) Graph showing the number of CD68 + cells in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. ( J ) Sagittal brain sections of the CD1 mice showing CD68 + (red) cells in the brain meninges of the EE VEH and EE ANA-12 mice. ( K ) Graph showing the number of fractones in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. Data are presented as the mean ± SEM, and n.s. = not statistically significant. * p value ≤ 0.05. ** p value ≤ 0.01. *** p value ≤ 0.001. In ( A , B , H , J ), the nuclei are in blue (TOPRO-3 nuclear staining). ( J ) is a single-plane confocal images. ( A , B , H ) are the maximum intensity projections of the z-stack confocal images. The scale bars represent 20 µm. White arrows indicate positive cells, while asterisks indicate fractones.
Anti Tubb3 Mouse Monoclonal Antibody, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Environmental Enrichment Induces Meningeal Niche Remodeling through TrkB-Mediated Signaling"

Article Title: Environmental Enrichment Induces Meningeal Niche Remodeling through TrkB-Mediated Signaling

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms221910657

ANA-12 administration reverts EE-induced changes in the meningeal niche. ( A ) Sagittal brain sections of the CD1 mice, showing GLAST + (red) cells above the meningeal basal lamina (green) in the EE VEH and EE ANA-12 mice. ( B ) Sagittal brain sections of the CD1 mice, showing β3-Tubulin + (red) cells above the meningeal basal lamina (green) in the EE VEH and EE ANA-12 mice. ( C ) Graph showing the number of GLAST + cells in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. ( D ) Graph showing the number of β3-Tubulin + cells in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. ( E ) Graph depicting gene expression analysis for Slc1a3 (GLAST) in the NO EE VEH, EE VEH and EE ANA-12 mice. Expression levels are reported as the log 2 fold change with respect to the NO EE VEH mice. ( F ) Graph depicting gene expression analysis for TUBB3 (β3-Tubulin) in the NO EE VEH, EE VEH and EE ANA-12 mice. Expression levels are reported as the log 2 fold change with respect to the NO EE VEH mice. ( G ) Representative western blot analysis of mouse brain meningeal protein extracts, which shows the presence of the DCX protein in the EE VEH, EE ANA-12 and NO EE VEH samples (left panel). The graph shows the differential fold change in protein expression for the DCX protein in the NO EE VEH, EE VEH and EE ANA-12 mice with respect to the NO EE VEH mice (right panel). ( H ) Sagittal brain sections of the CD1 mice, showing fractones (*) identified via laminin (green) staining in the brain meninges of EE VEH and EE ANA-12 mice. An insert illustrates laminin staining (fractones) below the meningeal nuclei (TOPRO-3). ( I ) Graph showing the number of CD68 + cells in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. ( J ) Sagittal brain sections of the CD1 mice showing CD68 + (red) cells in the brain meninges of the EE VEH and EE ANA-12 mice. ( K ) Graph showing the number of fractones in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. Data are presented as the mean ± SEM, and n.s. = not statistically significant. * p value ≤ 0.05. ** p value ≤ 0.01. *** p value ≤ 0.001. In ( A , B , H , J ), the nuclei are in blue (TOPRO-3 nuclear staining). ( J ) is a single-plane confocal images. ( A , B , H ) are the maximum intensity projections of the z-stack confocal images. The scale bars represent 20 µm. White arrows indicate positive cells, while asterisks indicate fractones.
Figure Legend Snippet: ANA-12 administration reverts EE-induced changes in the meningeal niche. ( A ) Sagittal brain sections of the CD1 mice, showing GLAST + (red) cells above the meningeal basal lamina (green) in the EE VEH and EE ANA-12 mice. ( B ) Sagittal brain sections of the CD1 mice, showing β3-Tubulin + (red) cells above the meningeal basal lamina (green) in the EE VEH and EE ANA-12 mice. ( C ) Graph showing the number of GLAST + cells in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. ( D ) Graph showing the number of β3-Tubulin + cells in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. ( E ) Graph depicting gene expression analysis for Slc1a3 (GLAST) in the NO EE VEH, EE VEH and EE ANA-12 mice. Expression levels are reported as the log 2 fold change with respect to the NO EE VEH mice. ( F ) Graph depicting gene expression analysis for TUBB3 (β3-Tubulin) in the NO EE VEH, EE VEH and EE ANA-12 mice. Expression levels are reported as the log 2 fold change with respect to the NO EE VEH mice. ( G ) Representative western blot analysis of mouse brain meningeal protein extracts, which shows the presence of the DCX protein in the EE VEH, EE ANA-12 and NO EE VEH samples (left panel). The graph shows the differential fold change in protein expression for the DCX protein in the NO EE VEH, EE VEH and EE ANA-12 mice with respect to the NO EE VEH mice (right panel). ( H ) Sagittal brain sections of the CD1 mice, showing fractones (*) identified via laminin (green) staining in the brain meninges of EE VEH and EE ANA-12 mice. An insert illustrates laminin staining (fractones) below the meningeal nuclei (TOPRO-3). ( I ) Graph showing the number of CD68 + cells in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. ( J ) Sagittal brain sections of the CD1 mice showing CD68 + (red) cells in the brain meninges of the EE VEH and EE ANA-12 mice. ( K ) Graph showing the number of fractones in 2 mm of the retrosplenial brain meninges of the NO EE VEH, EE VEH, EE ANA-12 and NO EE ANA-12 animals. Data are presented as the mean ± SEM, and n.s. = not statistically significant. * p value ≤ 0.05. ** p value ≤ 0.01. *** p value ≤ 0.001. In ( A , B , H , J ), the nuclei are in blue (TOPRO-3 nuclear staining). ( J ) is a single-plane confocal images. ( A , B , H ) are the maximum intensity projections of the z-stack confocal images. The scale bars represent 20 µm. White arrows indicate positive cells, while asterisks indicate fractones.

Techniques Used: Mouse Assay, Expressing, Western Blot, Staining

2) Product Images from "Inactivation of histone chaperone HIRA unmasks a link between normal embryonic development of melanoblasts and maintenance of adult melanocyte stem cells"

Article Title: Inactivation of histone chaperone HIRA unmasks a link between normal embryonic development of melanoblasts and maintenance of adult melanocyte stem cells

Journal: bioRxiv

doi: 10.1101/2022.04.22.489166

HIRA is required for melanoblast development. a, Scheme of single cell RNA sequencing method. 3 TyrCre::Hira wt/wt :tdTomato (WT) and 3 TyrCre::Hira fl/fl :tdTomato (KO) embryos were individually processed. b, t-SNE plots showing clusters based on gene expression among a total of 8655 single cells from 3 WT and 9743 single cells from 3 KO embryos. Cells from all 3 WT and 3 KO replicates are superimposed in each plot with the cells of each sample highlighted in a specific colour on each of the 6 plots. c, t-SNE plot of all 6 pooled samples showing 3 major cell types. d, t-SNE plots showing the expression of representative marker genes in the 3 major cell types. Expression level is color-coded in purple. e, 100% stacked column graph displaying the percentage of Sox10+ and mesenchymal cells within the tdTomato population in each of the 6 samples. Cell counts and percentages can be found in Supplementary Table 5. f, Scatter dot plots showing the percentages of cells in each of the populations in e in WT versus KO samples. g, t-SNE plot displaying Sox10 expression level in melanoblasts and glial cells h, Scatter dot plots displaying the average percentage of EdU positive nuclei in melb-a cells 10 days after infection with shHira1 and shHira2 lentiviral vectors, with shLuc as control. 3 independent replicates each (3 culture plates each each infected separately). i, Brightfield images of representative melb-a cells one week following infection as in ( h ). Uninfected (non-drug selected) parental cells are labelled as melb-a. Black arrows indicate enlarged cells (also traced in red) compared to parental and shLuc control (some cells traced in blue for comparison). Magnification 10x. j, Scatter dot plots representing the average nuclear area of cells in ( i ) as measured with ImageJ. k, Representative Western blots of HIRA, TUBB3 and the melanoblast-specific proteins c-KIT and DCT in lysates from shLuc, shHira1 and shHira2 melb-a cells, 3 independent replicates each, 10 days following infection and drug selection. GAPDH and Lamin A/C were used as loading and sample integrity controls, respectively. Blots performed in the same gel or a re-probed membrane are boxed together. Molecular weights were marked using Spectra multicolour broad range protein ladder. P: parental. l, c-KIT immunofluorescence (green) melb-a cells 10 days post-infection. m, TUBB3 immunofluorescence on same cells in ( l ). Scale bars: 25 μm. Scatter dot plot data in f , h and j were analysed using an unpaired t- test showing mean ± s.e.m. ns: non-significant (p > 0.05).
Figure Legend Snippet: HIRA is required for melanoblast development. a, Scheme of single cell RNA sequencing method. 3 TyrCre::Hira wt/wt :tdTomato (WT) and 3 TyrCre::Hira fl/fl :tdTomato (KO) embryos were individually processed. b, t-SNE plots showing clusters based on gene expression among a total of 8655 single cells from 3 WT and 9743 single cells from 3 KO embryos. Cells from all 3 WT and 3 KO replicates are superimposed in each plot with the cells of each sample highlighted in a specific colour on each of the 6 plots. c, t-SNE plot of all 6 pooled samples showing 3 major cell types. d, t-SNE plots showing the expression of representative marker genes in the 3 major cell types. Expression level is color-coded in purple. e, 100% stacked column graph displaying the percentage of Sox10+ and mesenchymal cells within the tdTomato population in each of the 6 samples. Cell counts and percentages can be found in Supplementary Table 5. f, Scatter dot plots showing the percentages of cells in each of the populations in e in WT versus KO samples. g, t-SNE plot displaying Sox10 expression level in melanoblasts and glial cells h, Scatter dot plots displaying the average percentage of EdU positive nuclei in melb-a cells 10 days after infection with shHira1 and shHira2 lentiviral vectors, with shLuc as control. 3 independent replicates each (3 culture plates each each infected separately). i, Brightfield images of representative melb-a cells one week following infection as in ( h ). Uninfected (non-drug selected) parental cells are labelled as melb-a. Black arrows indicate enlarged cells (also traced in red) compared to parental and shLuc control (some cells traced in blue for comparison). Magnification 10x. j, Scatter dot plots representing the average nuclear area of cells in ( i ) as measured with ImageJ. k, Representative Western blots of HIRA, TUBB3 and the melanoblast-specific proteins c-KIT and DCT in lysates from shLuc, shHira1 and shHira2 melb-a cells, 3 independent replicates each, 10 days following infection and drug selection. GAPDH and Lamin A/C were used as loading and sample integrity controls, respectively. Blots performed in the same gel or a re-probed membrane are boxed together. Molecular weights were marked using Spectra multicolour broad range protein ladder. P: parental. l, c-KIT immunofluorescence (green) melb-a cells 10 days post-infection. m, TUBB3 immunofluorescence on same cells in ( l ). Scale bars: 25 μm. Scatter dot plot data in f , h and j were analysed using an unpaired t- test showing mean ± s.e.m. ns: non-significant (p > 0.05).

Techniques Used: RNA Sequencing Assay, Expressing, Marker, Infection, Western Blot, Selection, Immunofluorescence

3) Product Images from "Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype"

Article Title: Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype

Journal: Autophagy

doi: 10.1080/15548627.2017.1395992

ALP inhibition enhances SNCA release. (A, B) ICC showing increased expression and homogeneous SNCA distribution in SNCA-overexpressing H4 cells (SNCA H4 compared to CTR H4) and neurons (SNCA-tg compared to WT). TUBB3 or GFP were used to visualize cellular outline. Scale bar: 10 µm. SNCA expression was also examined by WB analysis of cell lysate from H4 cells (C) or neurons (D). SNCA is not detectable in CTR H4, therefore quantification was not applicable. SNCA-tg neurons overexpress the protein by 2.5 fold compared to WT (*p = 0.024, N = 3). ACTB or TUBB3 were used as loading controls. (E) Dot blot analysis of SNCA H4 conditioned medium (CM) shows that relative total extracellular SNCA levels increase by treatment with the ALP inhibitors Baf and CQ over the corresponding Veh, which is plotted as a dotted line. (For Baf: *p
Figure Legend Snippet: ALP inhibition enhances SNCA release. (A, B) ICC showing increased expression and homogeneous SNCA distribution in SNCA-overexpressing H4 cells (SNCA H4 compared to CTR H4) and neurons (SNCA-tg compared to WT). TUBB3 or GFP were used to visualize cellular outline. Scale bar: 10 µm. SNCA expression was also examined by WB analysis of cell lysate from H4 cells (C) or neurons (D). SNCA is not detectable in CTR H4, therefore quantification was not applicable. SNCA-tg neurons overexpress the protein by 2.5 fold compared to WT (*p = 0.024, N = 3). ACTB or TUBB3 were used as loading controls. (E) Dot blot analysis of SNCA H4 conditioned medium (CM) shows that relative total extracellular SNCA levels increase by treatment with the ALP inhibitors Baf and CQ over the corresponding Veh, which is plotted as a dotted line. (For Baf: *p

Techniques Used: Inhibition, Immunocytochemistry, Expressing, Western Blot, Dot Blot

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    Promega ß tubulin iii
    Differentiation assays. (a) Left: Adipocyte with lipid vacuole resulted from adipogenic differentiation of CD146 positive cell stained with oil red. Right: Expression of PAPR‐γ2 and aP‐2 is shown following adipogenic differentiation using PCR. (b) Left: Mineralization and appropriate morphological changes are shown following osteogenic differentiation stained with alizarin red. Right: With osteogenic differentiation, expression of OPN and Col1α1 is revealed by PCR. (c) Left: With neurogenic differentiation typical dendritic cells which express appeared Right: <t>ß‐tubulin</t> III revealed by immune‐fluorescent staining. (d) Left: With hepatocytic differentiation, polygonal/flattened shape cells appeared at day 21 (differentiation step 2) Right: Hepatogenic differentiation was confirmed by qRT‐PCR as hepatogenic related genes were upregulated postdifferentiation, specially ALB and HNF with approximately 10‐ and 2.5‐fold higher expression after differentiation. The bars represent gene expressions before and after differentiation
    ß Tubulin Iii, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 2 article reviews
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    ß tubulin iii - by Bioz Stars, 2022-10
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    Differentiation assays. (a) Left: Adipocyte with lipid vacuole resulted from adipogenic differentiation of CD146 positive cell stained with oil red. Right: Expression of PAPR‐γ2 and aP‐2 is shown following adipogenic differentiation using PCR. (b) Left: Mineralization and appropriate morphological changes are shown following osteogenic differentiation stained with alizarin red. Right: With osteogenic differentiation, expression of OPN and Col1α1 is revealed by PCR. (c) Left: With neurogenic differentiation typical dendritic cells which express appeared Right: ß‐tubulin III revealed by immune‐fluorescent staining. (d) Left: With hepatocytic differentiation, polygonal/flattened shape cells appeared at day 21 (differentiation step 2) Right: Hepatogenic differentiation was confirmed by qRT‐PCR as hepatogenic related genes were upregulated postdifferentiation, specially ALB and HNF with approximately 10‐ and 2.5‐fold higher expression after differentiation. The bars represent gene expressions before and after differentiation

    Journal: Clinical and Experimental Dental Research

    Article Title: Differential expression of drug resistance genes in CD146 positive dental pulp derived stem cells and CD146 negative fibroblasts, et al. Differential expression of drug resistance genes in CD146 positive dental pulp derived stem cells and CD146 negative fibroblasts

    doi: 10.1002/cre2.297

    Figure Lengend Snippet: Differentiation assays. (a) Left: Adipocyte with lipid vacuole resulted from adipogenic differentiation of CD146 positive cell stained with oil red. Right: Expression of PAPR‐γ2 and aP‐2 is shown following adipogenic differentiation using PCR. (b) Left: Mineralization and appropriate morphological changes are shown following osteogenic differentiation stained with alizarin red. Right: With osteogenic differentiation, expression of OPN and Col1α1 is revealed by PCR. (c) Left: With neurogenic differentiation typical dendritic cells which express appeared Right: ß‐tubulin III revealed by immune‐fluorescent staining. (d) Left: With hepatocytic differentiation, polygonal/flattened shape cells appeared at day 21 (differentiation step 2) Right: Hepatogenic differentiation was confirmed by qRT‐PCR as hepatogenic related genes were upregulated postdifferentiation, specially ALB and HNF with approximately 10‐ and 2.5‐fold higher expression after differentiation. The bars represent gene expressions before and after differentiation

    Article Snippet: To confirm neural differentiation, immunostaining was performed for ß‐tubulin III (Promega cat number: G7121) as a specific antibody against neurons.

    Techniques: Staining, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR

    Effect of disease mutations on protein stability and protein-protein interactions. (a) Western blotting with anti-GFP antibody confirming the protein expression levels of wild-type Rrm2b, Actn2, Hprt1, Pnp, Tpk1, Gnmt, Gale, Fbp1, Klhl3, Tp53, Pnp, Smad4, and corresponding mutant alleles. β-tubulin and γ-tubulin were used as loading controls. Red denotes “interface residue” mutations, orange denotes “interface domain” mutations and blue denotes “away from the interface” mutations. (b) Likelihood of disruption of interactions by “interface residue”, “interface domain” and “away from the interface” mutations – overall and for stable mutants only; likelihood of a disease mutation disrupting a given interaction in the absence of structural information. Error bars indicate +SE. ( N = 204 mutations).

    Journal: PLoS Genetics

    Article Title: A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations

    doi: 10.1371/journal.pgen.1004819

    Figure Lengend Snippet: Effect of disease mutations on protein stability and protein-protein interactions. (a) Western blotting with anti-GFP antibody confirming the protein expression levels of wild-type Rrm2b, Actn2, Hprt1, Pnp, Tpk1, Gnmt, Gale, Fbp1, Klhl3, Tp53, Pnp, Smad4, and corresponding mutant alleles. β-tubulin and γ-tubulin were used as loading controls. Red denotes “interface residue” mutations, orange denotes “interface domain” mutations and blue denotes “away from the interface” mutations. (b) Likelihood of disruption of interactions by “interface residue”, “interface domain” and “away from the interface” mutations – overall and for stable mutants only; likelihood of a disease mutation disrupting a given interaction in the absence of structural information. Error bars indicate +SE. ( N = 204 mutations).

    Article Snippet: Anti-HA (Sigma H9658), anti-V5 (Invitrogen 46-0705), anti-β-tubulin (Promega G7121), and anti-GFP (Santa Cruz sc-9996) antibodies were used at 1∶3,000 dilutions for immunoblotting analysis.

    Techniques: Western Blot, Expressing, Mutagenesis

    ( A ) Representative pictures of differentiated neural stem cells, for neuronal ( β -III tubulin, green) and astrocyte (GFAP, red) Scale bars = 200 μ m ( B ) percentages of neuron, astrocyte after immunocytochemistry. IR = Immuno reactive, GFAP = glial fibrillary acidic protein. (Mean ± SEM; n = 3 independent experiments; * P

    Journal: Scientific Reports

    Article Title: An Experimental Investigation of Ultraweak Photon Emission from Adult Murine Neural Stem Cells

    doi: 10.1038/s41598-019-57352-4

    Figure Lengend Snippet: ( A ) Representative pictures of differentiated neural stem cells, for neuronal ( β -III tubulin, green) and astrocyte (GFAP, red) Scale bars = 200 μ m ( B ) percentages of neuron, astrocyte after immunocytochemistry. IR = Immuno reactive, GFAP = glial fibrillary acidic protein. (Mean ± SEM; n = 3 independent experiments; * P

    Article Snippet: The cells were washed 3X with PBS to remove PFA, incubated overnight at 4 °C with primary antibody neuronal marker, mouse monoclonal anti–III-tubulin (1:1000)(Promega, Madison, WI, USA), and glial marker, rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:500) (Dako Cytomation, Carpinteria, CA, USA) in PBST (PBS + 0.1% Triton-X) supplemented with 10% normal goat serum (NGS).

    Techniques: Immunocytochemistry

    Timeline of neuronal marker expression during reprogramming. Representative images of TUJ1 and MAP2 double immunostaining counterstained with DAPI (in blue) showing low levels of TUJ1 in dermal fibroblasts (in green), followed by intensification of expression at day 10, which is sustained until day 25 post-transduction with the U6.shREST.PGK.BRN2.PGK.ASCL1.WPRE construct. MAP2 expression (in red) is detectable in the nucleus at day 10 and is incrementally expressed in the processes from day 18 to 25. Scale bar = 25 μm.

    Journal: Frontiers in Neuroscience

    Article Title: Direct Neuronal Reprogramming for Disease Modeling Studies Using Patient-Derived Neurons: What Have We Learned?

    doi: 10.3389/fnins.2017.00530

    Figure Lengend Snippet: Timeline of neuronal marker expression during reprogramming. Representative images of TUJ1 and MAP2 double immunostaining counterstained with DAPI (in blue) showing low levels of TUJ1 in dermal fibroblasts (in green), followed by intensification of expression at day 10, which is sustained until day 25 post-transduction with the U6.shREST.PGK.BRN2.PGK.ASCL1.WPRE construct. MAP2 expression (in red) is detectable in the nucleus at day 10 and is incrementally expressed in the processes from day 18 to 25. Scale bar = 25 μm.

    Article Snippet: The following primary antibodies were used in the blocking solution overnight at 4°C: chicken anti-MAP2 (1:15,000; Abcam, ab5392); mouse anti-TUJ1 (1:1,000; Promega, G7121); rabbit anti-TUJ1 (1:1,000; BioLegend, 801201); chicken anti-VIM (1:5,000; Millipore, AB5733); mouse anti-TAU clone HT7 (1:500, Thermo Scientific, MN1000); mouse anti-TE7 (1:100, Millipore, CBL271).

    Techniques: Marker, Expressing, Double Immunostaining, Transduction, Construct

    Timeline of fibroblast marker expression during reprogramming. (A) Double immunofluorescent staining of the fibroblast and neuronal progenitor marker Vimentin (VIM; in green) and the neuronal marker TAU (in red) counterstained with DAPI (in blue) showing Vimentin expression in dermal fibroblasts as well as in a subpopulation of cells that are not expressing TAU at day 18 and 25 post-transduction. The majority of TAU expressing cells do not express VIM except for a few cells, which are double TAU/VIM+ (white arrowheads). (B) TUJ1 (cyan), MAP2 (red) and TE7 (green) triple immunostaining counterstained with DAPI (in blue) showing expression of the fibroblast marker TE7 in fibroblasts before transduction, whereas TUJ1+ and MAP2+ cells are negative for TE7, which is only detectable extracellularly at later time points during conversion. Scale bar in A = 25 μm, B = 100 μm.

    Journal: Frontiers in Neuroscience

    Article Title: Direct Neuronal Reprogramming for Disease Modeling Studies Using Patient-Derived Neurons: What Have We Learned?

    doi: 10.3389/fnins.2017.00530

    Figure Lengend Snippet: Timeline of fibroblast marker expression during reprogramming. (A) Double immunofluorescent staining of the fibroblast and neuronal progenitor marker Vimentin (VIM; in green) and the neuronal marker TAU (in red) counterstained with DAPI (in blue) showing Vimentin expression in dermal fibroblasts as well as in a subpopulation of cells that are not expressing TAU at day 18 and 25 post-transduction. The majority of TAU expressing cells do not express VIM except for a few cells, which are double TAU/VIM+ (white arrowheads). (B) TUJ1 (cyan), MAP2 (red) and TE7 (green) triple immunostaining counterstained with DAPI (in blue) showing expression of the fibroblast marker TE7 in fibroblasts before transduction, whereas TUJ1+ and MAP2+ cells are negative for TE7, which is only detectable extracellularly at later time points during conversion. Scale bar in A = 25 μm, B = 100 μm.

    Article Snippet: The following primary antibodies were used in the blocking solution overnight at 4°C: chicken anti-MAP2 (1:15,000; Abcam, ab5392); mouse anti-TUJ1 (1:1,000; Promega, G7121); rabbit anti-TUJ1 (1:1,000; BioLegend, 801201); chicken anti-VIM (1:5,000; Millipore, AB5733); mouse anti-TAU clone HT7 (1:500, Thermo Scientific, MN1000); mouse anti-TE7 (1:100, Millipore, CBL271).

    Techniques: Marker, Expressing, Staining, Transduction, Triple Immunostaining