mouse anti-brdu Search Results


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  • 94
    Thermo Fisher mouse anti brdu
    Mitochondrial replication. Cells with YFP-tagged mitochondria were grown in <t>BrdU</t> for 1–6 h, fixed, and Br-DNA <t>immunolabelled.</t> (A,B) On growth in BrdU, progressively more mitochondrial foci containing Br-DNA are seen. Bar: 2 μm. (C) Changes in the fraction of DNA foci containing Br-DNA (assuming cells contain 468 foci/cell; Table 1 , line 3); essentially all foci contain Br-DNA within 3 h. (D) Integrated intensity of Br-DNA labelling per focus (in arbitrary units, with the value at 1 h set to unity). The average intensity per focus is constant between 1 and 2 h, and then rises; extrapolating the line from 2–6 h to 22 h (the length of the cell cycle) gives an intensity of 10 (square). (E) Three models for replication. Two DNA foci, each initially containing 10 unreplicated genomes (open circles), are shown. (a) Here, a focus is a unit of replication. In the first hour, all genomes in one focus replicate together (red circles) and half the foci become labelled (as in Figure 6C ); in the second hour, all genomes in a second focus replicate together so that all genomes are now labelled (again as in Figure 6C ). However, during the third hour, the intensity of foci continues to increase (Figure 6D ), so we would have to assume that genomes re-replicate (dark red circles) and some would presumably have to be degraded to maintain genome numbers. (b) All genomes replicate independently of all others. The kinetics in (D) and (E) are consistent with this model. (c) Here, a polymerizing complex transfers from genome to genome in one focus before going to another focus; then genomes in one focus would replicate successively, before replication switched to another focus. This would give the progressive increase seen in Figure 6D , but many foci would remain unlabelled after 2 h (which is inconsistent with Figure 6E ).
    Mouse Anti Brdu, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mouse anti brdu
    Morphology of the <t>SOCS-2</t> −/− mouse pancreas, β-cell mass and growth hormone-induced proliferation of Ins-1E insulinoma cells in vitro. (A) Immunostainings with antibodies against the four major islet hormones were performed on paraffin sections from SOCS-2 −/− and wt pancreas. The proportions of the different cell types and the distribution of the cells within the islet are comparable in knockout and wt tissue. (B) pancreatic β-cell mass corrected for body weight was determined in 11–17 week old SOCS-2 −/− mice and wt controls (n = 6; male and female combined). No significant difference is seen between the two groups indicating a fully adapted β-cell mass in the knockout mice, which are generally bigger than their wt counterparts. (C) To determine whether SOCS-2 has a direct effect on growth hormone signaling in insulin-producing β-cells, Ins-1E cells were incubated for 24 hours with and without growth hormone (50 ng/ml; 2.2 nmol/l) in serum-free media. <t>BrdU</t> incorporation during that time period was quantified by immunostaining. Induction of proliferation by growth hormone is not significantly different in cells transfected with control or SOCS-2 specific siRNA (left) and also not in control and SOCS-2 overexpressing cells (right).
    Mouse Anti Brdu, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse anti brdu
    CAV1 is expressed by Layer Va CPN in an areally restricted fashion. A , A’ , Expression of CAV1 is areally restricted; CAV1 (green) is highly expressed by CPN (red; CTB 555 retrograde label) throughout primary and secondary somatosensory cortex (S1 and S2). CAV1 expression is not readily detected in CPN within motor cortex (M1), as delineated by Layer II/III expression of LMO4 (blue). A’ , Inset from A . Dashed line indicates M1–S1 boundary and cortical lamina. B , In S1, CAV1 (green) is largely excluded from the <t>CTIP2</t> (red)-expression domain (arrowheads) indicating SCPN, with only a small subpopulation of CTIP2+ve neurons extending into the CAV1-expression domain (arrow). C , In S2, the boundary between CAV1 (green) and CTIP2 (red) is not as clearly defined, with more CTIP2+ve neurons interspersed with CAV1 (arrows). Regions of higher magnification insets are indicated in B” and C’’ . D , At P6, CAV1 is expressed in Layer V, within the Satb2 and Bhlhb5-expressing domain, and below the serotonin-expressing barrel cortex in Layer IV. Regions of higher magnification insets are indicated in D’’ and D’’ . E , CAV1-expressing neurons are born between days E12.5 and E13.5 in both S1 and S2. deoxyuridine analogs (CldU or IdU) were injected at 12-h intervals throughout corticogenesis, and immunocytochemistry for <t>BrdU</t> (red) and CAV1 (green) was performed at P3, revealing that CAV1-expressing neocortical neurons, both in S1(somatosensory Layer V) and S2 (caudolateral expansion), are born between E12.3 and E13.5. Scale bars: 500 μm ( A’ ) and 100 μm ( B–E ).
    Mouse Anti Brdu, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 2381 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agilent technologies mouse anti brdu
    Akt2 deficiency is insufficient to inhibit the development of endometrial carcinoma in <t>Pten</t> +/– mice. ( a ) Histological sections representing the different grades of endometrial neopplasia in Pten +−/− mice (AH, atypical hyperplasia, CIS, carcinoma in situ ). The source of the tissue sections is indicated. ( b ) Incidence of endometrial neoplasia in Pten +/– , Pten +/– Akt2 –/– and wild-type mice. Total number of mice examined in each group is indicated in parentheses. ( c ) <t>BrdU</t> incorporation in the uteri of Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice in each group are indicated in parentheses. P -values are indicated.
    Mouse Anti Brdu, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti brdu antibody
    The receptor <t>RAGE</t> critically mediates the cellular response to S100A8/A9. a: Direct blockade by a specific RAGE blocking antibody reduces cellular proliferation. RAGE dependent proliferation was analyzed in normal primary, AK and SCC cells. Cells were incubated for 1 hour with a blocking anti-RAGE antibody (80μg/ml, as recommended by manufacturer) followed by S100A8/A9 stimulation for additional 24 hours. The differences in the proliferation after the blockade were assessed by <t>BrdU</t> incorporation (1 way Anova, Bonferroni`s Multiple test, **p
    Mouse Anti Brdu Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 687 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Developmental Studies Hybridoma Bank mouse anti brdu
    Temporal order of V0 excitatory neurogenesis. In all the experiments, fish in which a small number of V0 excitatory neurons expressed <t>GFP</t> were obtained by injecting the vglut2a :lDl-GFP DNA construct into Tg[ dbx1b :Cre] transgenic fish. A , Examples of three different time points. Top, 1.5 dpf. Two V0-eA neurons are labeled. Middle, 2.5 dpf. A V0-eB neuron is labeled (arrows indicate growing bifurcating axon). Bottom, A V0-eD neuron is labeled. B , Observation frequency of three classes of V0 excitatory neurons at three different time points. C , Schematics of <t>BrdU</t> incorporation experiments. BrdU was applied at a given time point, and embryos were incubated in the presence of BrdU until 72 hpf. Then, animals were moved to BrdU-free solution. At 120 hpf (5 dpf), neuronal morphology was examined. The animals were then processed for BrdU immunohistochemistry. D , Examples of V0-eB neurons. In each panel, the top images shows a stack of images from the living fish at 5 dpf, while the bottom three show images after BrdU immunohistochemistry. The GFP images are in green and the BrdU images are in red. Left, BrdU was applied at 24 hpf in this animal. The cell is positive for BrdU. Right, BrdU was applied at 36 hpf in this animal. The cell is negative for BrdU. E , Summary of BrdU incorporation experiments. Percentage of BrdU-positive and -negative cells for each cell type at a given time point of BrdU application is indicated. Scale bar, 20 μm.
    Mouse Anti Brdu, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam mouse anti brdu
    Neurogenesis and angiogenesis effects of human mesenchymal stem cells (hMSCs) in a stroke rat model. (A) Representative images of immunofluorescence staining with <t>anti-BrdU</t> <t>(red)/anti-doublecortin</t> (DCX; green) for analysis of neurogenesis. (B) Quantitative analysis of neurogenesis expressed as the proliferating cell number with anti-BrdU/anti-DCX positive cells in the subventricular zone. (C) Representative images of immunofluorescence staining with anti-vWF (red)/4’,6-diamidino-2-phenylindole (blue) for analysis of angiogenesis. (D) Quantitative analysis of angiogenesis expressed as the anti–von Willebrand factor positive area in the striatum. The quantitation of stained cells was performed in 6 fields per section. The data are presented as mean + SD (* P
    Mouse Anti Brdu, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare mouse anti brdu
    Premature postmitotic differentiation and depletion of proliferative neural precursor cells in the ventricular zone. A–R , Immunohistochemical analysis of SOX2 and <t>HUC/D</t> ( A–I ), SOX3 and HUC/D ( J–L ), and <t>BrdU</t> incorporation ( P–R ), as well as Hes5 nonradioactive in situ hybridization ( M–O ) on coronal midbrain sections of wild-type (WT), Fgfr1 cko ;Fgfr2 cko ( R1cko;R2cko ), and Fgfr1 cko ;Fgfr2 cko ;Fgfr3 null ( R1cko;R2cko;R3null ) embryos at E9.5–E11.5. S , The thickness of the ventricular zone (SOX2-positive) and the marginal zone (HUC/D-positive) was quantified (mean ± SD) at E11.5. G , The position at which layer thickness was measured is shown with white lines. For quantification of BrdU incorporation, the ventricular zone was divided into an approximately two-cell-layer-thick periventricular zone (zone1) and basal zone (zone2), visualized with broken lines ( P–R ). T , The proportion (mean ± SD) of BrdU-positive nuclei in zone1, zone2, and in the entire ventricular zone (total). White arrows point to decreased SOX2-positive layer and increased HUC/D-positive layer ( C , E , F , H , I ), downregulated SOX3 expression ( K , L ), and periventricular BrdU-positive cells ( Q , R ) in the mutants. Z1, Zone1; Z2, zone2. Asterisks indicate Hes5 -negative ventral domain. DAPI, 4′,6′-Diamidino-2-phenylindole. Scale bars, 100 μm. * p
    Mouse Anti Brdu, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 227 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse anti brdu
    Scatter plots display the correlations between the variables related with the hippocampal neuroplasticity and the hippocampal-dependent memory behavioral tests. Note: In the vmPFC HFS animals, the Morris water-maze target quadrant is positively correlated with the <t>Dcx</t> cell count ( A ), while the novel-object recognition task with no-HFS prior to testing shows positive correlation with the Dcx cell count ( C ) as well as the NeuN/Rbfox3 gene expression ( D ). No correlational association was found between the Morris water-maze and <t>BrdU</t> cell count after chronic vmPFC high-frequency stimulation ( B ). DOI: http://dx.doi.org/10.7554/eLife.04803.009
    Mouse Anti Brdu, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mouse anti brdu antibody
    Scatter plots display the correlations between the variables related with the hippocampal neuroplasticity and the hippocampal-dependent memory behavioral tests. Note: In the vmPFC HFS animals, the Morris water-maze target quadrant is positively correlated with the <t>Dcx</t> cell count ( A ), while the novel-object recognition task with no-HFS prior to testing shows positive correlation with the Dcx cell count ( C ) as well as the NeuN/Rbfox3 gene expression ( D ). No correlational association was found between the Morris water-maze and <t>BrdU</t> cell count after chronic vmPFC high-frequency stimulation ( B ). DOI: http://dx.doi.org/10.7554/eLife.04803.009
    Mouse Anti Brdu Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Mitochondrial replication. Cells with YFP-tagged mitochondria were grown in BrdU for 1–6 h, fixed, and Br-DNA immunolabelled. (A,B) On growth in BrdU, progressively more mitochondrial foci containing Br-DNA are seen. Bar: 2 μm. (C) Changes in the fraction of DNA foci containing Br-DNA (assuming cells contain 468 foci/cell; Table 1 , line 3); essentially all foci contain Br-DNA within 3 h. (D) Integrated intensity of Br-DNA labelling per focus (in arbitrary units, with the value at 1 h set to unity). The average intensity per focus is constant between 1 and 2 h, and then rises; extrapolating the line from 2–6 h to 22 h (the length of the cell cycle) gives an intensity of 10 (square). (E) Three models for replication. Two DNA foci, each initially containing 10 unreplicated genomes (open circles), are shown. (a) Here, a focus is a unit of replication. In the first hour, all genomes in one focus replicate together (red circles) and half the foci become labelled (as in Figure 6C ); in the second hour, all genomes in a second focus replicate together so that all genomes are now labelled (again as in Figure 6C ). However, during the third hour, the intensity of foci continues to increase (Figure 6D ), so we would have to assume that genomes re-replicate (dark red circles) and some would presumably have to be degraded to maintain genome numbers. (b) All genomes replicate independently of all others. The kinetics in (D) and (E) are consistent with this model. (c) Here, a polymerizing complex transfers from genome to genome in one focus before going to another focus; then genomes in one focus would replicate successively, before replication switched to another focus. This would give the progressive increase seen in Figure 6D , but many foci would remain unlabelled after 2 h (which is inconsistent with Figure 6E ).

    Journal: BMC Biology

    Article Title: The functional organization of mitochondrial genomes in human cells

    doi: 10.1186/1741-7007-2-9

    Figure Lengend Snippet: Mitochondrial replication. Cells with YFP-tagged mitochondria were grown in BrdU for 1–6 h, fixed, and Br-DNA immunolabelled. (A,B) On growth in BrdU, progressively more mitochondrial foci containing Br-DNA are seen. Bar: 2 μm. (C) Changes in the fraction of DNA foci containing Br-DNA (assuming cells contain 468 foci/cell; Table 1 , line 3); essentially all foci contain Br-DNA within 3 h. (D) Integrated intensity of Br-DNA labelling per focus (in arbitrary units, with the value at 1 h set to unity). The average intensity per focus is constant between 1 and 2 h, and then rises; extrapolating the line from 2–6 h to 22 h (the length of the cell cycle) gives an intensity of 10 (square). (E) Three models for replication. Two DNA foci, each initially containing 10 unreplicated genomes (open circles), are shown. (a) Here, a focus is a unit of replication. In the first hour, all genomes in one focus replicate together (red circles) and half the foci become labelled (as in Figure 6C ); in the second hour, all genomes in a second focus replicate together so that all genomes are now labelled (again as in Figure 6C ). However, during the third hour, the intensity of foci continues to increase (Figure 6D ), so we would have to assume that genomes re-replicate (dark red circles) and some would presumably have to be degraded to maintain genome numbers. (b) All genomes replicate independently of all others. The kinetics in (D) and (E) are consistent with this model. (c) Here, a polymerizing complex transfers from genome to genome in one focus before going to another focus; then genomes in one focus would replicate successively, before replication switched to another focus. This would give the progressive increase seen in Figure 6D , but many foci would remain unlabelled after 2 h (which is inconsistent with Figure 6E ).

    Article Snippet: Visualizing newly-made transcripts Cells on coverslips were grown [ ] in 2.5 mM bromouridine (BrU; Sigma-Aldrich), fixed (20 min; 4°C) in 4% paraformaldehyde in 250 mM HEPES (pH 7.4), and Br-RNA indirectly immunolabelled using mouse anti-BrdU (Caltag) and donkey anti-mouse Fc conjugated with Cy3.

    Techniques:

    Morphology of the SOCS-2 −/− mouse pancreas, β-cell mass and growth hormone-induced proliferation of Ins-1E insulinoma cells in vitro. (A) Immunostainings with antibodies against the four major islet hormones were performed on paraffin sections from SOCS-2 −/− and wt pancreas. The proportions of the different cell types and the distribution of the cells within the islet are comparable in knockout and wt tissue. (B) pancreatic β-cell mass corrected for body weight was determined in 11–17 week old SOCS-2 −/− mice and wt controls (n = 6; male and female combined). No significant difference is seen between the two groups indicating a fully adapted β-cell mass in the knockout mice, which are generally bigger than their wt counterparts. (C) To determine whether SOCS-2 has a direct effect on growth hormone signaling in insulin-producing β-cells, Ins-1E cells were incubated for 24 hours with and without growth hormone (50 ng/ml; 2.2 nmol/l) in serum-free media. BrdU incorporation during that time period was quantified by immunostaining. Induction of proliferation by growth hormone is not significantly different in cells transfected with control or SOCS-2 specific siRNA (left) and also not in control and SOCS-2 overexpressing cells (right).

    Journal: Islets

    Article Title: No non-redundant function of suppressor of cytokine signaling 2 in insulin producing ?-cells

    doi: 10.4161/isl.2.4.12556

    Figure Lengend Snippet: Morphology of the SOCS-2 −/− mouse pancreas, β-cell mass and growth hormone-induced proliferation of Ins-1E insulinoma cells in vitro. (A) Immunostainings with antibodies against the four major islet hormones were performed on paraffin sections from SOCS-2 −/− and wt pancreas. The proportions of the different cell types and the distribution of the cells within the islet are comparable in knockout and wt tissue. (B) pancreatic β-cell mass corrected for body weight was determined in 11–17 week old SOCS-2 −/− mice and wt controls (n = 6; male and female combined). No significant difference is seen between the two groups indicating a fully adapted β-cell mass in the knockout mice, which are generally bigger than their wt counterparts. (C) To determine whether SOCS-2 has a direct effect on growth hormone signaling in insulin-producing β-cells, Ins-1E cells were incubated for 24 hours with and without growth hormone (50 ng/ml; 2.2 nmol/l) in serum-free media. BrdU incorporation during that time period was quantified by immunostaining. Induction of proliferation by growth hormone is not significantly different in cells transfected with control or SOCS-2 specific siRNA (left) and also not in control and SOCS-2 overexpressing cells (right).

    Article Snippet: The following antibodies were used in this study: Rabbit anti-SOCS-2 (1:500; Abcam), Mouse anti-β-actin (1:10,000, Sigma), Mouse anti-BrdU (1:1,000, Sigma), Guinea pig anti-insulin (1:1,000, Dako), Rabbit anti-glucagon (1:500, Chemicon); Rabbit anti-somatostatin (1:100, Chemicon); Rabbit anti-pancreatic polypeptide (1:30, Chemicon).

    Techniques: In Vitro, Knock-Out, Mouse Assay, Incubation, BrdU Incorporation Assay, Immunostaining, Transfection

    SmgGDS-558 physically interacts with UBF, and this interaction promotes the nucleolar accumulation of SmgGDS-558. ( a ) HEK293T cells were transfected with cDNAs encoding the HA vector or HA-tagged SmgGDS, followed by immunoprecipitation using HA antibody and silver staining to detect co-precipitating proteins. Mass spectrometry identified UBF as one of the co-precipitating proteins (HC and LC; heavy and light chains, respectively, of antibodies used in the immunoprecipitation). ( b ) Lysates from HEK293T cells transfected with cDNAs encoding the HA vector or HA-tagged SmgGDS were immunoprecipitated using HA antibody, followed by immunoblotting using antibodies to UBF and HA ( n =3). ( c ) HEK293T cells were transfected with cDNAs encoding the HA vector or SmgGDS-558-NES mut -HA along with non-targeting (NT) siRNA or UBF siRNA. After 72 h, the cells were immunofluorescently stained with HA antibody, UBF antibody and 4,6-diamidino-2-phenylindole (DAPI), and examined by confocal fluorescence microscopy ( n =3). ( d ) HEK293T cells expressing SmgGDS-558-NES mut -HA were treated with or without the RNA Pol I inhibitor CX-5461 (1 μ m , 2 h), followed by FuRD (2 m m , 15 min), and immunofluorescently stained using antibodies to HA and BrdU ( n =3). Images were obtained by confocal microscopy. ( e ) NCI-H1703 cells were transfected with the indicated siRNAs to deplete SmgGDS, and 72 h later quantitative PCR was conducted to examine 47S pre-rRNA levels (normalized to cellular GAPDH). Control cells were treated with CX-5461 (1 μ m , 2 h) before collecting RNA. Error bars represent ±s.e.m. of three biological replicates, and statistical significance was determined by one-way analysis of variance and Holm-Sidak multiple comparisons test (* P

    Journal: Oncogene

    Article Title: SmgGDS is a transient nucleolar protein that protects cells from nucleolar stress and promotes the cell cycle by regulating DREAM complex gene expression

    doi: 10.1038/onc.2017.280

    Figure Lengend Snippet: SmgGDS-558 physically interacts with UBF, and this interaction promotes the nucleolar accumulation of SmgGDS-558. ( a ) HEK293T cells were transfected with cDNAs encoding the HA vector or HA-tagged SmgGDS, followed by immunoprecipitation using HA antibody and silver staining to detect co-precipitating proteins. Mass spectrometry identified UBF as one of the co-precipitating proteins (HC and LC; heavy and light chains, respectively, of antibodies used in the immunoprecipitation). ( b ) Lysates from HEK293T cells transfected with cDNAs encoding the HA vector or HA-tagged SmgGDS were immunoprecipitated using HA antibody, followed by immunoblotting using antibodies to UBF and HA ( n =3). ( c ) HEK293T cells were transfected with cDNAs encoding the HA vector or SmgGDS-558-NES mut -HA along with non-targeting (NT) siRNA or UBF siRNA. After 72 h, the cells were immunofluorescently stained with HA antibody, UBF antibody and 4,6-diamidino-2-phenylindole (DAPI), and examined by confocal fluorescence microscopy ( n =3). ( d ) HEK293T cells expressing SmgGDS-558-NES mut -HA were treated with or without the RNA Pol I inhibitor CX-5461 (1 μ m , 2 h), followed by FuRD (2 m m , 15 min), and immunofluorescently stained using antibodies to HA and BrdU ( n =3). Images were obtained by confocal microscopy. ( e ) NCI-H1703 cells were transfected with the indicated siRNAs to deplete SmgGDS, and 72 h later quantitative PCR was conducted to examine 47S pre-rRNA levels (normalized to cellular GAPDH). Control cells were treated with CX-5461 (1 μ m , 2 h) before collecting RNA. Error bars represent ±s.e.m. of three biological replicates, and statistical significance was determined by one-way analysis of variance and Holm-Sidak multiple comparisons test (* P

    Article Snippet: For FuRD labeling, cells were incubated with FuRD (2 mm , 15 min, 37ºC) before fixing and staining with mouse anti-BrdU antibody (Sigma, B8434), as described above.

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Silver Staining, Mass Spectrometry, Staining, Fluorescence, Microscopy, Expressing, Confocal Microscopy, Real-time Polymerase Chain Reaction

    Altered expression of p27Kip1 and Sox2 in Eya1 bor/bor cochleae. ( A – F ) Immunostaining of E14.0 (A– I ) and E18.5 (G– L ) cochlear sections with an anti-p27KIP1 (A–C, J–L), anti-BrdU (G–I) or anti-Sox2 (D–F,

    Journal:

    Article Title: Eya1 gene dosage critically affects the development of sensory epithelia in the mammalian inner ear

    doi: 10.1093/hmg/ddn229

    Figure Lengend Snippet: Altered expression of p27Kip1 and Sox2 in Eya1 bor/bor cochleae. ( A – F ) Immunostaining of E14.0 (A– I ) and E18.5 (G– L ) cochlear sections with an anti-p27KIP1 (A–C, J–L), anti-BrdU (G–I) or anti-Sox2 (D–F,

    Article Snippet: For immunohistochemistry, primary antibodies were specific for MYO7A (Proteus Biosciences INC), p27KIP1 (BD Biosciences pharmingen), SOX2 (Chemicon), β-galactosidase (Sigma), Jag1 (Santa Cruz) and mouse monoclonal anti-BrdU (Sigma).

    Techniques: Expressing, Immunostaining

    CCN1 inhibits hepatocyte DNA synthesis through integrin α 6 and p53 A, freshly isolated hepatocytes from adult Ccn1 wt/wt , Ccn1 ΔHep and Ccn1 dm/dm mice were incubated with purified wild type, DM, and D125A CCN1 proteins (10 μg/ml each) with or without EGF (0.1 μg/ml) at 37°C for 2 days, and BrdU incorporation determined. Percentage of BrdU-positive cells were counted in 5 random HPF and presented as mean ± s.d. of triplicate determinations. Experiments were repeated three times with similar results. B, hepatocytes from Ccn1 wt/wt mice were incubated with siRNAs targeting integrin α 6 or α v subunits, or a non-targeting control, and treated with CCN1, EGF, or both, and BrdU incorporation assessed as above. Knockdown was confirmed by qPCR of the target integrin mRNA (right). C, p53 and EGFR expression was knocked-down by siRNAs in hepatocytes (inset), and cells were further incubated with CCN1 with or without EGF, and BrdU incorporation assayed as above.

    Journal: Oncogene

    Article Title: The Matricellular Protein CCN1 Suppresses Hepatocarcinogenesis by Inhibiting Compensatory Proliferation

    doi: 10.1038/onc.2015.190

    Figure Lengend Snippet: CCN1 inhibits hepatocyte DNA synthesis through integrin α 6 and p53 A, freshly isolated hepatocytes from adult Ccn1 wt/wt , Ccn1 ΔHep and Ccn1 dm/dm mice were incubated with purified wild type, DM, and D125A CCN1 proteins (10 μg/ml each) with or without EGF (0.1 μg/ml) at 37°C for 2 days, and BrdU incorporation determined. Percentage of BrdU-positive cells were counted in 5 random HPF and presented as mean ± s.d. of triplicate determinations. Experiments were repeated three times with similar results. B, hepatocytes from Ccn1 wt/wt mice were incubated with siRNAs targeting integrin α 6 or α v subunits, or a non-targeting control, and treated with CCN1, EGF, or both, and BrdU incorporation assessed as above. Knockdown was confirmed by qPCR of the target integrin mRNA (right). C, p53 and EGFR expression was knocked-down by siRNAs in hepatocytes (inset), and cells were further incubated with CCN1 with or without EGF, and BrdU incorporation assayed as above.

    Article Snippet: For BrdU incorporation assay, cells were incubated with 0.1 mM BrdU at 37°C for 3 hrs, fixed and stained with mouse anti-BrdU mAb (clone BU-1, Millipore #MAB3510).

    Techniques: DNA Synthesis, Isolation, Mouse Assay, Incubation, Purification, BrdU Incorporation Assay, Real-time Polymerase Chain Reaction, Expressing

    Pronephric epithelial proliferation. (A–C,E,F) Red: anti-BrdU staining, Green: anti-GFP staining. BrdU incorporation was measured for 24 h, between 48 and 72 hpf. (A) Proliferation in the proximal tubule is localized to the neck region (arrow). (B) Proliferation in the anterior part of the distal tubule is low. (C) Proliferation in the ret1 -positive pronephric duct segment is high. (D) Cartoon depicting zebrafish pronephros. The proximal tubule (ET33d10 GFP positive domain) and the pronephric duct ( ret1 :GFP positive domain) are shaded black. The glomerulus and the distal tubule (ET11-9 GFP positive domain) are not shaded. (E) Proliferation in the posterior proximal tubule is low. (F) Proliferation in the posterior distal tubule is high. (G) Quantification of pronephric epithelial proliferation after 24 hour BrdU incorporation. The length of the tubule is plotted on the horizontal axis (measured from the cloaca). The linear density of BrdU positive nuclei (per 100 µm tubule length) is plotted on the vertical axis. Squares: pronephric proliferation between 3 and 4 dpf (n = 3). Circles: proliferation between 1 and 2 dpf (n = 3). See also Figure S1 .

    Journal: PLoS ONE

    Article Title: Mechanical Stretch and PI3K Signaling Link Cell Migration and Proliferation to Coordinate Epithelial Tubule Morphogenesis in the Zebrafish Pronephros

    doi: 10.1371/journal.pone.0039992

    Figure Lengend Snippet: Pronephric epithelial proliferation. (A–C,E,F) Red: anti-BrdU staining, Green: anti-GFP staining. BrdU incorporation was measured for 24 h, between 48 and 72 hpf. (A) Proliferation in the proximal tubule is localized to the neck region (arrow). (B) Proliferation in the anterior part of the distal tubule is low. (C) Proliferation in the ret1 -positive pronephric duct segment is high. (D) Cartoon depicting zebrafish pronephros. The proximal tubule (ET33d10 GFP positive domain) and the pronephric duct ( ret1 :GFP positive domain) are shaded black. The glomerulus and the distal tubule (ET11-9 GFP positive domain) are not shaded. (E) Proliferation in the posterior proximal tubule is low. (F) Proliferation in the posterior distal tubule is high. (G) Quantification of pronephric epithelial proliferation after 24 hour BrdU incorporation. The length of the tubule is plotted on the horizontal axis (measured from the cloaca). The linear density of BrdU positive nuclei (per 100 µm tubule length) is plotted on the vertical axis. Squares: pronephric proliferation between 3 and 4 dpf (n = 3). Circles: proliferation between 1 and 2 dpf (n = 3). See also Figure S1 .

    Article Snippet: The antibodies used were: Anti- BrdU mouse monoclonal antibody (clone BU-133, Sigma), anti-GFP rabbit polyclonal antibody (Sigma G1544), anti- phospho histone H3 rabbit monoclonal antibody (clone MC463, Millipore), alpha6F mouse monoclonal antibody (Developmental Studies Hybridoma Bank), and anti-cadherin 17 rabbit polyclonal antibody (Gift from Dr. Zhaoxia Sun, Yale University).

    Techniques: BrdU Staining, Staining, BrdU Incorporation Assay

    Collective epithelial migration stimulates cell proliferation in the distal tubule. (A, B) Cell proliferation in the distal tubule (ET11-9 GFP domain) after anterior obstruction. Obstruction was induced at 30 hpf, BrdU incorporation was assessed between 2 and 3 dpf. (A) Total number of BrdU+ nuclei in the distal ET11-9 domain. White bar: control (n = 4), black bar: anterior obstruction (n = 4). P = 0.01. (B) Spatial distribution of the BrdU-positive nuclei per 100 µm length of the distal tubule (measured from the posterior border of the ET11-9 domain). Squares: control (n = 4), Circles: anterior obstruction (n = 4). (C,D) Cell proliferation in the distal nephron in mindbomb mutants. mindbomb ( mib ) heterozygotes were in-crossed and injected with tnnt2 morpholino to control for vascular defects in mib mutants. BrdU incorporation was assessed between 2 and 3 dpf. Homozygous mib mutants were separated from their siblings based on their axis curvature phenotype. (E) Total amount of BrdU incorporation in pronephric duct (black bar: control, n = 8; dark-grey bar: mindbomb , n = 8; P > 0.05) and in the posterior distal tubule (light grey bar: control, n = 8; white bar: mindbomb , n = 8; P

    Journal: PLoS ONE

    Article Title: Mechanical Stretch and PI3K Signaling Link Cell Migration and Proliferation to Coordinate Epithelial Tubule Morphogenesis in the Zebrafish Pronephros

    doi: 10.1371/journal.pone.0039992

    Figure Lengend Snippet: Collective epithelial migration stimulates cell proliferation in the distal tubule. (A, B) Cell proliferation in the distal tubule (ET11-9 GFP domain) after anterior obstruction. Obstruction was induced at 30 hpf, BrdU incorporation was assessed between 2 and 3 dpf. (A) Total number of BrdU+ nuclei in the distal ET11-9 domain. White bar: control (n = 4), black bar: anterior obstruction (n = 4). P = 0.01. (B) Spatial distribution of the BrdU-positive nuclei per 100 µm length of the distal tubule (measured from the posterior border of the ET11-9 domain). Squares: control (n = 4), Circles: anterior obstruction (n = 4). (C,D) Cell proliferation in the distal nephron in mindbomb mutants. mindbomb ( mib ) heterozygotes were in-crossed and injected with tnnt2 morpholino to control for vascular defects in mib mutants. BrdU incorporation was assessed between 2 and 3 dpf. Homozygous mib mutants were separated from their siblings based on their axis curvature phenotype. (E) Total amount of BrdU incorporation in pronephric duct (black bar: control, n = 8; dark-grey bar: mindbomb , n = 8; P > 0.05) and in the posterior distal tubule (light grey bar: control, n = 8; white bar: mindbomb , n = 8; P

    Article Snippet: The antibodies used were: Anti- BrdU mouse monoclonal antibody (clone BU-133, Sigma), anti-GFP rabbit polyclonal antibody (Sigma G1544), anti- phospho histone H3 rabbit monoclonal antibody (clone MC463, Millipore), alpha6F mouse monoclonal antibody (Developmental Studies Hybridoma Bank), and anti-cadherin 17 rabbit polyclonal antibody (Gift from Dr. Zhaoxia Sun, Yale University).

    Techniques: Migration, BrdU Incorporation Assay, Injection

    Distal proliferation supports prolonged epithelial migration. (A–D) 1.5 µm confocal slices of the distal tubule. Green: GFP (ET11-9 GFP transgenic in A–B, Tg(atp1a1a.4:GFP) transgenic in C D). Red: BrdU. Magenta: DAPI. (A) BrdU incorporation in the distal tubule (2–3 dpf) in control fish. (B) Lack of BrdU incorporation in distal tubule between (2–3 dpf) when embryo is treated with 30 µM LY294002. Cells outside of the kidney continued to incorporate BrdU. (C) DAPI staining of distal tubule in control fish (4 dpf). (D) DAPI staining of distal tubule treated with 30 µM LY294002 (4 dpf). The distal tubule was markedly thinned in LY294002 condition (B, D). (E) estimated cross-sectional tubule area based on measured maximal diameter of the tubule in confocal stacks at 3 dpf. Circles: control (n = 5). Squares: LY294002 treated fish (n = 3). (F) Linear nuclear density (DAPI) in control fish (circles, n = 3) and LY294002 treated fish (squares, n = 3) confirmed linear stretching of the distal tubule in Ly294002 treated fish (4 dpf). (G, H) Kidney segment lengths in (G) control fish and (H) LY294002 treated fish at 4dpf. ET11-9 transgenic fish were used to estimate the segment lengths: ‘a’, ‘b’ and ‘c’ represent proximal tubule, distal tubule and the pronephric duct. (I–N) Individual frames of time lapse movies of the actively migrating pronephric epithelia in the presence of 30 µM LY294002. Arrowheads point to the individual traced cells. Time lapse immediately after addition of LY294002 (I, J), 24 hours after addition of LY294002 (K, L), and 48 hours after addition of LY294002 (M, N). Frame pairs in I and J, K and L, and M and N are separated by 6 hours. (O) Average epithelial migration rates after different durations of LY294002 exposure. Each bar represents average migration rate of 4 different individual cells in the proximal tubule (ET11-9:GFP). (P) Lengths of proximal tubule (designated ‘a’ in G), distal tubule (designated ‘b’ in G) and pronephric duct (designated ‘c’ in G) in control 96 hpf fish and fish treated with 30 µM LY294002 starting at 30 hpf. White bars: control (n = 9), black bars: LY294002 (n = 9). See also Figure S2 .

    Journal: PLoS ONE

    Article Title: Mechanical Stretch and PI3K Signaling Link Cell Migration and Proliferation to Coordinate Epithelial Tubule Morphogenesis in the Zebrafish Pronephros

    doi: 10.1371/journal.pone.0039992

    Figure Lengend Snippet: Distal proliferation supports prolonged epithelial migration. (A–D) 1.5 µm confocal slices of the distal tubule. Green: GFP (ET11-9 GFP transgenic in A–B, Tg(atp1a1a.4:GFP) transgenic in C D). Red: BrdU. Magenta: DAPI. (A) BrdU incorporation in the distal tubule (2–3 dpf) in control fish. (B) Lack of BrdU incorporation in distal tubule between (2–3 dpf) when embryo is treated with 30 µM LY294002. Cells outside of the kidney continued to incorporate BrdU. (C) DAPI staining of distal tubule in control fish (4 dpf). (D) DAPI staining of distal tubule treated with 30 µM LY294002 (4 dpf). The distal tubule was markedly thinned in LY294002 condition (B, D). (E) estimated cross-sectional tubule area based on measured maximal diameter of the tubule in confocal stacks at 3 dpf. Circles: control (n = 5). Squares: LY294002 treated fish (n = 3). (F) Linear nuclear density (DAPI) in control fish (circles, n = 3) and LY294002 treated fish (squares, n = 3) confirmed linear stretching of the distal tubule in Ly294002 treated fish (4 dpf). (G, H) Kidney segment lengths in (G) control fish and (H) LY294002 treated fish at 4dpf. ET11-9 transgenic fish were used to estimate the segment lengths: ‘a’, ‘b’ and ‘c’ represent proximal tubule, distal tubule and the pronephric duct. (I–N) Individual frames of time lapse movies of the actively migrating pronephric epithelia in the presence of 30 µM LY294002. Arrowheads point to the individual traced cells. Time lapse immediately after addition of LY294002 (I, J), 24 hours after addition of LY294002 (K, L), and 48 hours after addition of LY294002 (M, N). Frame pairs in I and J, K and L, and M and N are separated by 6 hours. (O) Average epithelial migration rates after different durations of LY294002 exposure. Each bar represents average migration rate of 4 different individual cells in the proximal tubule (ET11-9:GFP). (P) Lengths of proximal tubule (designated ‘a’ in G), distal tubule (designated ‘b’ in G) and pronephric duct (designated ‘c’ in G) in control 96 hpf fish and fish treated with 30 µM LY294002 starting at 30 hpf. White bars: control (n = 9), black bars: LY294002 (n = 9). See also Figure S2 .

    Article Snippet: The antibodies used were: Anti- BrdU mouse monoclonal antibody (clone BU-133, Sigma), anti-GFP rabbit polyclonal antibody (Sigma G1544), anti- phospho histone H3 rabbit monoclonal antibody (clone MC463, Millipore), alpha6F mouse monoclonal antibody (Developmental Studies Hybridoma Bank), and anti-cadherin 17 rabbit polyclonal antibody (Gift from Dr. Zhaoxia Sun, Yale University).

    Techniques: Migration, Transgenic Assay, BrdU Incorporation Assay, Fluorescence In Situ Hybridization, Staining

    CAV1 is expressed by Layer Va CPN in an areally restricted fashion. A , A’ , Expression of CAV1 is areally restricted; CAV1 (green) is highly expressed by CPN (red; CTB 555 retrograde label) throughout primary and secondary somatosensory cortex (S1 and S2). CAV1 expression is not readily detected in CPN within motor cortex (M1), as delineated by Layer II/III expression of LMO4 (blue). A’ , Inset from A . Dashed line indicates M1–S1 boundary and cortical lamina. B , In S1, CAV1 (green) is largely excluded from the CTIP2 (red)-expression domain (arrowheads) indicating SCPN, with only a small subpopulation of CTIP2+ve neurons extending into the CAV1-expression domain (arrow). C , In S2, the boundary between CAV1 (green) and CTIP2 (red) is not as clearly defined, with more CTIP2+ve neurons interspersed with CAV1 (arrows). Regions of higher magnification insets are indicated in B” and C’’ . D , At P6, CAV1 is expressed in Layer V, within the Satb2 and Bhlhb5-expressing domain, and below the serotonin-expressing barrel cortex in Layer IV. Regions of higher magnification insets are indicated in D’’ and D’’ . E , CAV1-expressing neurons are born between days E12.5 and E13.5 in both S1 and S2. deoxyuridine analogs (CldU or IdU) were injected at 12-h intervals throughout corticogenesis, and immunocytochemistry for BrdU (red) and CAV1 (green) was performed at P3, revealing that CAV1-expressing neocortical neurons, both in S1(somatosensory Layer V) and S2 (caudolateral expansion), are born between E12.3 and E13.5. Scale bars: 500 μm ( A’ ) and 100 μm ( B–E ).

    Journal: eNeuro

    Article Title: Caveolin1 Identifies a Specific Subpopulation of Cerebral Cortex Callosal Projection Neurons (CPN) Including Dual Projecting Cortical Callosal/Frontal Projection Neurons (CPN/FPN)

    doi: 10.1523/ENEURO.0234-17.2017

    Figure Lengend Snippet: CAV1 is expressed by Layer Va CPN in an areally restricted fashion. A , A’ , Expression of CAV1 is areally restricted; CAV1 (green) is highly expressed by CPN (red; CTB 555 retrograde label) throughout primary and secondary somatosensory cortex (S1 and S2). CAV1 expression is not readily detected in CPN within motor cortex (M1), as delineated by Layer II/III expression of LMO4 (blue). A’ , Inset from A . Dashed line indicates M1–S1 boundary and cortical lamina. B , In S1, CAV1 (green) is largely excluded from the CTIP2 (red)-expression domain (arrowheads) indicating SCPN, with only a small subpopulation of CTIP2+ve neurons extending into the CAV1-expression domain (arrow). C , In S2, the boundary between CAV1 (green) and CTIP2 (red) is not as clearly defined, with more CTIP2+ve neurons interspersed with CAV1 (arrows). Regions of higher magnification insets are indicated in B” and C’’ . D , At P6, CAV1 is expressed in Layer V, within the Satb2 and Bhlhb5-expressing domain, and below the serotonin-expressing barrel cortex in Layer IV. Regions of higher magnification insets are indicated in D’’ and D’’ . E , CAV1-expressing neurons are born between days E12.5 and E13.5 in both S1 and S2. deoxyuridine analogs (CldU or IdU) were injected at 12-h intervals throughout corticogenesis, and immunocytochemistry for BrdU (red) and CAV1 (green) was performed at P3, revealing that CAV1-expressing neocortical neurons, both in S1(somatosensory Layer V) and S2 (caudolateral expansion), are born between E12.3 and E13.5. Scale bars: 500 μm ( A’ ) and 100 μm ( B–E ).

    Article Snippet: Primary antibodies and dilutions were used as follows: rabbit anti-CAV1, 1:500 (Cell Signaling #3238; RRID: AB_2072166 ); goat anti-LMO4, 1:200 (Santa Cruz Biotechnology SC- 11122; RRID: AB_648429 ); rat anti-CTIP2 1:500 (Abcam ab18465; RRID: AB_2064130 ), mouse anti-BrdU, 1:500 (Becton Dickinson #347580; clone B44; RRID: AB_10015219 ; detects IdU); rat anti-BrdU, 1:500 (Accurate #OBT- 0030; clone BU1/75; RRID: AB_2313756 ; detects CldU); rabbit anti-GFP, 1:500 (Invitrogen; RRID: AB_221569 ); mouse anti-SATB2, 1:500 (Abcam ab51502; RRID: AB_882455 ); goat anti-BHLHB5, 1:200 (Santa Cruz Biotechnology SC-6045; RRID: AB_2065343 ); and rabbit anti-5-HT, 1:1000 (Immunostar 20080; RRID: AB_572263 ).

    Techniques: Expressing, CtB Assay, Injection, Immunocytochemistry

    sap1-1 impacts DNA replication. ( A ) FACS profile of DNA content in WT and sap1-1 cells. Cells carrying the cdc10-v50 mutation were arrested in G1 and then released. Numbers on the left indicate the time in minutes after the release. ( B ) DNA replication profile in WT and sap1-1 cells. The differences in BrdU incorporation were plotted across subtelomeric domains and at certain chromosome arm regions that gained association with Bqt3 in sap1-1 cells. GFP-Bqt3 ChIP enrichment in WT and sap1-1 is shown on the top. Orange and blue circles represent early- and late-replication origins, respectively. ( C ) Heat maps of Chr.1 for both WT and sap1-1 cells showing the Mcm6 ChIP signal around replication origins (10-kb regions). ( D ) Rad11-GFP forms discrete foci in sap1-1 cells. Cells expressing Rad11-GFP (green) and Hht1(histone H3)-RFP (red) were grown at 33 °C for 6 h. The percentage of mononuclear cells with Rad11-GFP foci ( n = 84 for WT and n = 103 for sap1-1 cells) is shown. ( E ) Mitotic cells expressing Rad11-GFP (green) and Hht1-RFP (red). sap1-1 cells enter mitotic nuclear division in the presence of Rad11 foci. Chromatin (Hht1-RFP) appears fragmented and lags during the M-phase in sap1-1 cells. Rad11-GFP forms a fine bridge between the daughter nuclei and is enriched near fragmented chromatin masses. (Scale bar, 5 μm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Shelterin components mediate genome reorganization in response to replication stress

    doi: 10.1073/pnas.1705527114

    Figure Lengend Snippet: sap1-1 impacts DNA replication. ( A ) FACS profile of DNA content in WT and sap1-1 cells. Cells carrying the cdc10-v50 mutation were arrested in G1 and then released. Numbers on the left indicate the time in minutes after the release. ( B ) DNA replication profile in WT and sap1-1 cells. The differences in BrdU incorporation were plotted across subtelomeric domains and at certain chromosome arm regions that gained association with Bqt3 in sap1-1 cells. GFP-Bqt3 ChIP enrichment in WT and sap1-1 is shown on the top. Orange and blue circles represent early- and late-replication origins, respectively. ( C ) Heat maps of Chr.1 for both WT and sap1-1 cells showing the Mcm6 ChIP signal around replication origins (10-kb regions). ( D ) Rad11-GFP forms discrete foci in sap1-1 cells. Cells expressing Rad11-GFP (green) and Hht1(histone H3)-RFP (red) were grown at 33 °C for 6 h. The percentage of mononuclear cells with Rad11-GFP foci ( n = 84 for WT and n = 103 for sap1-1 cells) is shown. ( E ) Mitotic cells expressing Rad11-GFP (green) and Hht1-RFP (red). sap1-1 cells enter mitotic nuclear division in the presence of Rad11 foci. Chromatin (Hht1-RFP) appears fragmented and lags during the M-phase in sap1-1 cells. Rad11-GFP forms a fine bridge between the daughter nuclei and is enriched near fragmented chromatin masses. (Scale bar, 5 μm.)

    Article Snippet: The cdc10-v50 strains carrying the herpes simplex virus thymidine kinase expression module Pnmt1-TK and the human nucleoside transporter module Padh1-hENT were grown at 35 °C for 4 h and were released from the G1 block by transfer to medium supplemented with 200 μM BrdU and 10 mM HU at 26 °C for 90 min. BrdU-labeled DNA was extracted and used for immunoprecipitation with mouse anti-BrdU antibody (BD Pharmingen).

    Techniques: FACS, Mutagenesis, BrdU Incorporation Assay, Chromatin Immunoprecipitation, Expressing

    Replication profiles of WT and sap1-1 cells. ( A ) Replication profile of a segment of chromosome 1 in WT cells. Detection of early-replication origins in the cdc10-v50 single-mutant background by BrdU immunoprecipitation. Cells were released from the G1-phase in the presence of HU. ( B ) Replication profiles of subtelomeric regions in WT ( Top ) and sap1-1 ( Middle ) cells. Cells carrying the cdc10-v50 allele were arrested and released from G1-phase in the presence of HU. ( Bottom ) The difference between sap1-1 ). ( C ) Mcm6 ChIP-chip analysis of chromosome 1 in WT and sap1-1 cells. ( D ) DNA replication profile of chromosome arm regions in WT and sap1-1 cells. GFP-Bqt3 and Mcm6 ChIP-chip are shown in the top and bottom graphs, respectively. IP, immunoprecipitation.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Shelterin components mediate genome reorganization in response to replication stress

    doi: 10.1073/pnas.1705527114

    Figure Lengend Snippet: Replication profiles of WT and sap1-1 cells. ( A ) Replication profile of a segment of chromosome 1 in WT cells. Detection of early-replication origins in the cdc10-v50 single-mutant background by BrdU immunoprecipitation. Cells were released from the G1-phase in the presence of HU. ( B ) Replication profiles of subtelomeric regions in WT ( Top ) and sap1-1 ( Middle ) cells. Cells carrying the cdc10-v50 allele were arrested and released from G1-phase in the presence of HU. ( Bottom ) The difference between sap1-1 ). ( C ) Mcm6 ChIP-chip analysis of chromosome 1 in WT and sap1-1 cells. ( D ) DNA replication profile of chromosome arm regions in WT and sap1-1 cells. GFP-Bqt3 and Mcm6 ChIP-chip are shown in the top and bottom graphs, respectively. IP, immunoprecipitation.

    Article Snippet: The cdc10-v50 strains carrying the herpes simplex virus thymidine kinase expression module Pnmt1-TK and the human nucleoside transporter module Padh1-hENT were grown at 35 °C for 4 h and were released from the G1 block by transfer to medium supplemented with 200 μM BrdU and 10 mM HU at 26 °C for 90 min. BrdU-labeled DNA was extracted and used for immunoprecipitation with mouse anti-BrdU antibody (BD Pharmingen).

    Techniques: Mutagenesis, Immunoprecipitation, Chromatin Immunoprecipitation

    Skeletal muscle morphology 1 week after trauma. Red color represents the nuclear Hoechst staining. Green color refers to BrdU staining. Co-localization of the signals ( yellow color ) represents the newly divided cells. Blue color refers to laminin-staining. Three different regions can be identified: intact muscle, a necrotic core, and a penumbra which is characterized by disturbed myofibers and a high percentage of proliferating cells

    Journal: European Journal of Trauma and Emergency Surgery

    Article Title: Muscle regeneration is undisturbed by repeated polytraumatic injury

    doi: 10.1007/s00068-010-0034-9

    Figure Lengend Snippet: Skeletal muscle morphology 1 week after trauma. Red color represents the nuclear Hoechst staining. Green color refers to BrdU staining. Co-localization of the signals ( yellow color ) represents the newly divided cells. Blue color refers to laminin-staining. Three different regions can be identified: intact muscle, a necrotic core, and a penumbra which is characterized by disturbed myofibers and a high percentage of proliferating cells

    Article Snippet: After blocking in 1.5% normal goat serum, a mouse monoclonal anti-BrdU antibody (BD Pharmingen, #555627) and rabbit anti-laminin (Sigma, #L9393) were applied at 1:200 dilution.

    Techniques: Staining, BrdU Staining

    Newly formed muscle fiber with BrDU positive nuclei. Five weeks after the trauma, clear laminin staining and completely regenerated muscular structure can be investigated. Arrows show BrDU positive nuclei incorporated into the myofiber

    Journal: European Journal of Trauma and Emergency Surgery

    Article Title: Muscle regeneration is undisturbed by repeated polytraumatic injury

    doi: 10.1007/s00068-010-0034-9

    Figure Lengend Snippet: Newly formed muscle fiber with BrDU positive nuclei. Five weeks after the trauma, clear laminin staining and completely regenerated muscular structure can be investigated. Arrows show BrDU positive nuclei incorporated into the myofiber

    Article Snippet: After blocking in 1.5% normal goat serum, a mouse monoclonal anti-BrdU antibody (BD Pharmingen, #555627) and rabbit anti-laminin (Sigma, #L9393) were applied at 1:200 dilution.

    Techniques: Staining

    Shh-mediated RPC proliferation and cell fate specification requires Hes1 . (a–c) In vivo anti-pH3 staining of the central retina adjacent to the optic nerve (asterisks) in P5 wild-type (Wt), PtchlacZ +/− , and PtchlacZ +/− Hes1 +/− retinas. Arrows indicate pH3-positive cells. Note that pH3+ cells in the vicinity of the optic nerve are rare in Wt and compound heterozygous mice. Bar, 100 μm. (d) Quantitative analysis of BrdU incorporation in vivo from P5 Wt ( n = 3), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 3), and PtchlacZ +/− Hes1 +/− ( n = 6) retinas. Values represent the mean number of BrdU-positive cells counted from three sections per animal. (e) Quantification of the proportion of BrdU + cells in single-cell dissociates from the retinas of Wt ( n = 5), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 8), and PtchlacZ +/− Hes1 +/− ( n = 7) retinas at P5. (f) Retinal explants from Hes1 −/− ( n = 3) or Wt ( n = 3) animals were treated with a Smo agonist for 3 d, dissociated, and scored for the proportion of BrdU + DAPI + cells. (g) Quantitative analysis for BrdU, CRALBP, Chx10, rhodopsin, and recoverin-positive cells in Smo agonist–treated P0 retinal explants electroporated with GFP and Hes1DN. Values are based on scoring marker+ cells among the transfected cohort in dissociates from retinal explants and represent the fold induction of double-positive (marker+GFP+) cells in GFP + Ag and Hes1DN + Ag cultures compared with double-positive cells in GFP-transfected untreated explants. There is no difference in proliferation or cell type composition in GFP and Hes1DN-transfected cells in untreated explants. Error bars represent SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity

    doi: 10.1083/jcb.200805155

    Figure Lengend Snippet: Shh-mediated RPC proliferation and cell fate specification requires Hes1 . (a–c) In vivo anti-pH3 staining of the central retina adjacent to the optic nerve (asterisks) in P5 wild-type (Wt), PtchlacZ +/− , and PtchlacZ +/− Hes1 +/− retinas. Arrows indicate pH3-positive cells. Note that pH3+ cells in the vicinity of the optic nerve are rare in Wt and compound heterozygous mice. Bar, 100 μm. (d) Quantitative analysis of BrdU incorporation in vivo from P5 Wt ( n = 3), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 3), and PtchlacZ +/− Hes1 +/− ( n = 6) retinas. Values represent the mean number of BrdU-positive cells counted from three sections per animal. (e) Quantification of the proportion of BrdU + cells in single-cell dissociates from the retinas of Wt ( n = 5), Hes1 +/− ( n = 3), PtchlacZ +/− ( n = 8), and PtchlacZ +/− Hes1 +/− ( n = 7) retinas at P5. (f) Retinal explants from Hes1 −/− ( n = 3) or Wt ( n = 3) animals were treated with a Smo agonist for 3 d, dissociated, and scored for the proportion of BrdU + DAPI + cells. (g) Quantitative analysis for BrdU, CRALBP, Chx10, rhodopsin, and recoverin-positive cells in Smo agonist–treated P0 retinal explants electroporated with GFP and Hes1DN. Values are based on scoring marker+ cells among the transfected cohort in dissociates from retinal explants and represent the fold induction of double-positive (marker+GFP+) cells in GFP + Ag and Hes1DN + Ag cultures compared with double-positive cells in GFP-transfected untreated explants. There is no difference in proliferation or cell type composition in GFP and Hes1DN-transfected cells in untreated explants. Error bars represent SEM. *, P

    Article Snippet: Antibodies used in this study include rabbit polyclonal anti-CRALBP (a kind gift from J. Saari, University of Washington, Seattle, WA), mouse monoclonal anti-BrdU (BD), mouse monoclonal anti-rhodopsin , rabbit polyclonal anti-recoverin (Millipore), sheep polyclonal anti-Chx10 (a gift from R. Bremner, Toronto Western Research Institute, Toronto, Ontario, Canada), rabbit polyclonal phosphohistone H3 (Millipore), and rabbit polyclonal anti-GFP (Invitrogen).

    Techniques: In Vivo, Staining, Mouse Assay, BrdU Incorporation Assay, Marker, Transfection

    Gli2 is required for the Shh effects on proliferation and cell fate. Retinal explants were cultured from wild-type (Wt; n = 3) and Gli2 −/− ( n = 3) mice at E18 for 3 d in culture with or without a Smo agonist. IHC was performed on dissociated cells using anti-BrdU, anti-CRALBP, anti-rhodopsin, and anti-recoverin antibodies. Values represent the fold induction of positive cells in Wt + Ag or Gli2 −/− + Ag cultures compared with nontreated explants. Error bars represent SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity

    doi: 10.1083/jcb.200805155

    Figure Lengend Snippet: Gli2 is required for the Shh effects on proliferation and cell fate. Retinal explants were cultured from wild-type (Wt; n = 3) and Gli2 −/− ( n = 3) mice at E18 for 3 d in culture with or without a Smo agonist. IHC was performed on dissociated cells using anti-BrdU, anti-CRALBP, anti-rhodopsin, and anti-recoverin antibodies. Values represent the fold induction of positive cells in Wt + Ag or Gli2 −/− + Ag cultures compared with nontreated explants. Error bars represent SEM. *, P

    Article Snippet: Antibodies used in this study include rabbit polyclonal anti-CRALBP (a kind gift from J. Saari, University of Washington, Seattle, WA), mouse monoclonal anti-BrdU (BD), mouse monoclonal anti-rhodopsin , rabbit polyclonal anti-recoverin (Millipore), sheep polyclonal anti-Chx10 (a gift from R. Bremner, Toronto Western Research Institute, Toronto, Ontario, Canada), rabbit polyclonal phosphohistone H3 (Millipore), and rabbit polyclonal anti-GFP (Invitrogen).

    Techniques: Cell Culture, Mouse Assay, Immunohistochemistry

    Visualization and characterization of viral DNA RC in KSHV-infected DMVEC and TPA-induced JSC-1 PEL cells. (a to d) Double-label IFA detection of ORF-K8 accumulated in RC at 72 h after TPA treatment of JSC-1 PEL cells using FITC anti-K8 PAb and rhodamine anti-PPF MAb. (e to h) Double-label IFA detection of K8 colocalized with newly synthesized DNA in TPA-treated PEL cell RC after a 30-min BrdU pulse-label experiment using rhodamine anti-K8 PAb and FITC anti-BrdU MAb. (i to l) Double-label IFA detection of replication compartments surrounded by PODs in TPA-treated PEL cells, using rhodamine-labeled anti-PPF MAb and FITC-labeled anti-PML PAb. (m to o) Double-label IFA detection of K8 accumulated in RC formed in KSHV-infected DMVEC, using rhodamine anti-K8 PAb and FITC anti-BrdU MAb (30 min of BrdU pulse-labeling). (p to r) Double-label IFA detection of PPF in infected DMVEC RC surrounded by PODs, using rhodamine anti-PPF MAb and FITC anti-PML PAb.

    Journal: Journal of Virology

    Article Title: Origin-Independent Assembly of Kaposi's Sarcoma-Associated Herpesvirus DNA Replication Compartments in Transient Cotransfection Assays and Association with the ORF-K8 Protein and Cellular PML

    doi: 10.1128/JVI.75.3.1487-1506.2001

    Figure Lengend Snippet: Visualization and characterization of viral DNA RC in KSHV-infected DMVEC and TPA-induced JSC-1 PEL cells. (a to d) Double-label IFA detection of ORF-K8 accumulated in RC at 72 h after TPA treatment of JSC-1 PEL cells using FITC anti-K8 PAb and rhodamine anti-PPF MAb. (e to h) Double-label IFA detection of K8 colocalized with newly synthesized DNA in TPA-treated PEL cell RC after a 30-min BrdU pulse-label experiment using rhodamine anti-K8 PAb and FITC anti-BrdU MAb. (i to l) Double-label IFA detection of replication compartments surrounded by PODs in TPA-treated PEL cells, using rhodamine-labeled anti-PPF MAb and FITC-labeled anti-PML PAb. (m to o) Double-label IFA detection of K8 accumulated in RC formed in KSHV-infected DMVEC, using rhodamine anti-K8 PAb and FITC anti-BrdU MAb (30 min of BrdU pulse-labeling). (p to r) Double-label IFA detection of PPF in infected DMVEC RC surrounded by PODs, using rhodamine anti-PPF MAb and FITC anti-PML PAb.

    Article Snippet: Antibodies used included mouse anti-BrdU MAb (Becton Dickinson), rabbit anti-ORF6 PAb (SSB), mouse anti-ORF59 MAb (PPF), rabbit polyclonal anti-K8, rabbit polyclonal anti-PML(C), directed against amino acid positions 484 to 498 of the human 90-kDa PML isoform , mouse MAb and rabbit PAb anti-Flag (Sigma), and mouse MAb and rabbit PAb anti-Myc (Santa Cruz).

    Techniques: Infection, Immunofluorescence, Synthesized, Labeling

    All six core KSHV DNA replication proteins are required for the assembly of complete RC-like structures in contransfected Vero cells, and active DNA synthesis occurs within KSHV RC assembled in the presence of EBV ori-Lyt and ZTA. (a to f) Double-label IFA demonstrating colocalization of POL, PRI, and PAF with SSB in large pseudo-RC in transient assembly assays. SSB was detected by IFA with FITC-labeled anti-SSB rabbit PAb, whereas POL, PRI, and PAF Flag-tagged fusion proteins were detected with rhodamine-labeled anti-Flag mouse MAb. (a and b) Flag-tagged POL cotransfected with the untagged plasmids encoding PPF, PRI, PAF, HEL, and SSB; (c and d) Flag-tagged PRI cotransfected with all five other untagged plasmids; (e and f) Flag-tagged PAF cotransfected with all five other untagged plasmids. The incompletely transfected cell at the upper right in panels c and d displays nuclear diffuse SSB and cytoplasmic PRI. Deliberate omission of any of the six replication genes also disrupted RC formation (not shown). (g to j) Evidence for newly synthesized DNA within KSHV RC formed in Vero cells cotransfected with the complete set of untagged KSHV core replication expression plasmids (POL, PPF, PRI, PAF, HEL, and SSB) plus EBV ori-Lyt and the ZTA DNA binding protein. Cells were pulse labeled for 30 min with BrdU prior to double-label screening for RC formation with an anti-SSB PAb and for active DNA synthesis with an anti-BrdU MAb. (g and i) Anti-KSHV SSB antibody to identify intranuclear RC containing core viral DNA replication proteins. (h and j) Anti-BrdU antibody to identify sites of ongoing DNA synthesis in the same cells (arrows indicate structures that resemble complete viral DNA RC). The anti-BrdU antibody also detected typical background speckled BrdU incorporation patterns found in the 25% of untransfected cells in S phase.

    Journal: Journal of Virology

    Article Title: Origin-Independent Assembly of Kaposi's Sarcoma-Associated Herpesvirus DNA Replication Compartments in Transient Cotransfection Assays and Association with the ORF-K8 Protein and Cellular PML

    doi: 10.1128/JVI.75.3.1487-1506.2001

    Figure Lengend Snippet: All six core KSHV DNA replication proteins are required for the assembly of complete RC-like structures in contransfected Vero cells, and active DNA synthesis occurs within KSHV RC assembled in the presence of EBV ori-Lyt and ZTA. (a to f) Double-label IFA demonstrating colocalization of POL, PRI, and PAF with SSB in large pseudo-RC in transient assembly assays. SSB was detected by IFA with FITC-labeled anti-SSB rabbit PAb, whereas POL, PRI, and PAF Flag-tagged fusion proteins were detected with rhodamine-labeled anti-Flag mouse MAb. (a and b) Flag-tagged POL cotransfected with the untagged plasmids encoding PPF, PRI, PAF, HEL, and SSB; (c and d) Flag-tagged PRI cotransfected with all five other untagged plasmids; (e and f) Flag-tagged PAF cotransfected with all five other untagged plasmids. The incompletely transfected cell at the upper right in panels c and d displays nuclear diffuse SSB and cytoplasmic PRI. Deliberate omission of any of the six replication genes also disrupted RC formation (not shown). (g to j) Evidence for newly synthesized DNA within KSHV RC formed in Vero cells cotransfected with the complete set of untagged KSHV core replication expression plasmids (POL, PPF, PRI, PAF, HEL, and SSB) plus EBV ori-Lyt and the ZTA DNA binding protein. Cells were pulse labeled for 30 min with BrdU prior to double-label screening for RC formation with an anti-SSB PAb and for active DNA synthesis with an anti-BrdU MAb. (g and i) Anti-KSHV SSB antibody to identify intranuclear RC containing core viral DNA replication proteins. (h and j) Anti-BrdU antibody to identify sites of ongoing DNA synthesis in the same cells (arrows indicate structures that resemble complete viral DNA RC). The anti-BrdU antibody also detected typical background speckled BrdU incorporation patterns found in the 25% of untransfected cells in S phase.

    Article Snippet: Antibodies used included mouse anti-BrdU MAb (Becton Dickinson), rabbit anti-ORF6 PAb (SSB), mouse anti-ORF59 MAb (PPF), rabbit polyclonal anti-K8, rabbit polyclonal anti-PML(C), directed against amino acid positions 484 to 498 of the human 90-kDa PML isoform , mouse MAb and rabbit PAb anti-Flag (Sigma), and mouse MAb and rabbit PAb anti-Myc (Santa Cruz).

    Techniques: DNA Synthesis, Immunofluorescence, Labeling, Transfection, Synthesized, Expressing, Binding Assay, BrdU Incorporation Assay

    A reduced DG is associated with a drop in neonatal but not adult neurogenesis (A, B) Proliferating cells labeled with immunohistochemistry for BrdU (arrows in A), or PH3 in 8-week old brains. BrdU-IR cells appear equally dense in the SGZ and hilus in control and double mutant brains (A). Rectangles in (A) indicate field size used for counting BrdU-IR and PH3-IR cells. (B) Density of PH3-IR cells in the SGZ of control and double mutant brains (n=3, each group) is not significantly different. (C) Coronal sections through the DG hilus at P5 processed with double immunofluorescence for BrdU and the neuronal marker NeuN. Double immunofluorescence (merge) labels twice as many newborn neurons in the DG hilus of a control mouse than a double mutant (white arrows indicate double-positive cells). (D) Density of PH3-IR cells in the hilus of neonatal control and double mutant brains (n=3, each group). The density of mitotic cells was significantly less in double mutants (p=0.003, one tailed t-test). Data represented as means +/− sem. Scale bar in (C) = 100 μm in (A); 50 μm in (C).

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior

    doi: 10.1523/JNEUROSCI.0550-10.2010

    Figure Lengend Snippet: A reduced DG is associated with a drop in neonatal but not adult neurogenesis (A, B) Proliferating cells labeled with immunohistochemistry for BrdU (arrows in A), or PH3 in 8-week old brains. BrdU-IR cells appear equally dense in the SGZ and hilus in control and double mutant brains (A). Rectangles in (A) indicate field size used for counting BrdU-IR and PH3-IR cells. (B) Density of PH3-IR cells in the SGZ of control and double mutant brains (n=3, each group) is not significantly different. (C) Coronal sections through the DG hilus at P5 processed with double immunofluorescence for BrdU and the neuronal marker NeuN. Double immunofluorescence (merge) labels twice as many newborn neurons in the DG hilus of a control mouse than a double mutant (white arrows indicate double-positive cells). (D) Density of PH3-IR cells in the hilus of neonatal control and double mutant brains (n=3, each group). The density of mitotic cells was significantly less in double mutants (p=0.003, one tailed t-test). Data represented as means +/− sem. Scale bar in (C) = 100 μm in (A); 50 μm in (C).

    Article Snippet: For immunohistochemistry, primary antibodies were: anti-phospho Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428) rabbit polyclonal antibody (1:50, Cell Signaling Technology), anti-phospho Histone H3 (Ser10) rabbit polyclonal antibody (1:200, Upstate), anti-Calbindin D-28K rabbit polyclonal antibody (1:250, Chemicon), anti-Neuronal Nuclei (NeuN) mouse monoclonal antibody (1:10, Millipore), anti-BrdU mouse monoclonal antibody (1:75, Becton Dickinson) for IHC and anti-BrdU mouse monoclonal antibody (1:50, Serotec) for immunofluorescence, anti-Tyrosine Hydroxylase (TH) antibody 1:5000 (Beckton Dickinson), anti-β-tubulin (Tuj1) (1:500, Covance), anti-Axin2 rabbit polyclonal antibody (1:5000, Abcam), anti-Olig2 rabbit polyclonal antibody (1:1000, Abcam), anti-(cleaved) Caspase3 rabbit polyclonal antibody (1:200, Cell Signaling), anti-DKK1 rabbit polyclonal antibody (1:2000, Santa Cruz Biotechnology), anti-Calretinin rabbit polyclonal antibody (1:1000, Millipore) and anti-Acvr1 (ALK2) rabbit polyclonal antibody (1:2000, Novus Biologicals).

    Techniques: Labeling, Immunohistochemistry, Mutagenesis, Immunofluorescence, Marker, One-tailed Test

    SMAD1 is necessary for the formation of mDA neurons. Representative midbrain coronal sections of WT and Smad1 Nes mutant littermates at E12.5 and E14.5. phospho-SMAD-1/5/8-positive cells (arrowheads) are strongly reduced in Smad1 Nes mutants ( A , A′ ). Midbrain TH + neurons are significantly reduced in Smad1 Nes mutants ( B – B

    Journal: The Journal of Neuroscience

    Article Title: BMP/SMAD Pathway Promotes Neurogenesis of Midbrain Dopaminergic Neurons In Vivo and in Human Induced Pluripotent and Neural Stem Cells

    doi: 10.1523/JNEUROSCI.1540-17.2018

    Figure Lengend Snippet: SMAD1 is necessary for the formation of mDA neurons. Representative midbrain coronal sections of WT and Smad1 Nes mutant littermates at E12.5 and E14.5. phospho-SMAD-1/5/8-positive cells (arrowheads) are strongly reduced in Smad1 Nes mutants ( A , A′ ). Midbrain TH + neurons are significantly reduced in Smad1 Nes mutants ( B – B" ). NURR1 postmitotic neurons appear reduced in Smad1 Nes mutants ( C , C′ ), while the numbers of LMX1a + mDA progenitors ( D – D" ) and POU1F4 + red nucleus progenitors ( E – E" ) show no statistical changes between phenotypes. In mDA progenitor domain of Smad1 Nes mutants, the number of PH3 + mitotic cells flanked by NKX6.1 expression ( F – F" ) and LMX1A + KI67 + cells ( G – G" ) are significantly increased. The quitting fraction calculated as a ratio of cells that have exited cell cycle (LMX1A + BrdU + KI67 − ) and cells that are actively cycling (LMX1A + BrdU + KI67 + ) is significantly reduced in Smad1 Nes knock-outs ( H – H" ). There is no difference in the numbers of LMX1A + NGN2 + cells between genotypes ( I – I" ). In Smad1 Nes mutants, BETA-catenin is normally expressed ( J – J′ ). SHH has retracted in both WT and mutants from the midline at this developmental stage, as measured by comparing the signal intensity of the midline to the adjacent basal plate ( K – K′ ). Also at E14.5, the number of TH + cells is decreased in Smad1 Nes mutants. The reduction in TH + PITX3 + cells exceeds the reduction observed in TH + cells ( L – L"′ ). Two tailed unpaired Student, t test * p

    Article Snippet: Primary antibodies used for the in vivo study were as follows: mouse anti-SHH (1:10), mouse anti-NKX6.1 (1:10), mouse anti-ISLET1/2 (1:10), mouse anti-MSX1/2 (1:10), mouse anti-FOXA2 (1:10), mouse anti-NESTIN (1:10), mouse anti-EN1 (1:50) - all from DSHB, rabbit anti-LMX1A (1:400, catalog #AB10533; Millipore), rabbit anti-LMX1B (1:1000, gift from Dr. C. Birchmeier, MDC, Berlin), rabbit anti-NURR1 (1:100, catalog #SC991; Santa Cruz Biotechnology), mouse anti-NURR1 (1:100, catalog #376984; Santa Cruz Biotechnology) mouse anti-neurogenin-2 (1:800, catalog #MAB3314; R & D Systems), rabbit anti-TH (1:200, catalog #AB152; Millipore), mouse anti-TH (1:200, catalog #MAB318; Millipore), rabbit anti-phospho-SMAD1/5/8 (1:200, #9511S Cell Signaling), sheep anti-BMP5 (1:10, catalog #AF6176; R & D Systems), rabbit anti-phospho-histone-H3 (1:1000, catalog #06-570; Millipore), rabbit anti-phospho-P38 (1:1000, catalog #4511 Cell Signaling), rabbit anti-GIRK2 (1:200, catalog #APC-006; Alomone Laboratories), rabbit anti-SOX6 (1:500, catalog #ab30455; Abcam), mouse anti-POU4F1 (1:300, catalog #sc8429; Santa Cruz Biotechnology), mouse anti-N-cadherin (1:200, catalog #610920; BD Biosciences), rabbit anti-ZO1 (1:100, catalog #40-2200; Invitrogen), rabbit anti-KI67 (1:100, catalog # ab16667; Abcam), rabbit anti-cleaved-caspase 3 (1:100, catalog #3661; Cell Signaling Technology), rabbit anti-MAP-2 (1:200, catalog #sc20172; Santa Cruz Biotechnology), rabbit anti-β-catenin (1:400, catalog #9587s; Cell Signaling Technology), rabbit anti-SHH (1:50, catalog #sc9024; Santa Cruz Biotechnology), rabbit anti-BMPR1B (1:100, catalog #10537; Orbigen), rabbit anti-PITX3 (catalog #38-2850; Thermo Fisher Scientific), mouse anti-BrdU (catalog #B2531; Sigma-Aldrich), rabbit anti-CCND1(1:150, catalog #sc-450; Santa Cruz Biotechnology), rabbit anti-phospho-β-catenin (1:200, catalog #9561s; Cell Signaling Technology), rabbit anti-calbindin (1:500, catalog # d -28k; Swant), and rabbit anti-β III tubulin/TUJ1 (1:500, catalog #302 302; Synaptic Systems).

    Techniques: Multiple Displacement Amplification, Mutagenesis, Expressing, Two Tailed Test

    Akt2 deficiency is insufficient to inhibit the development of endometrial carcinoma in Pten +/– mice. ( a ) Histological sections representing the different grades of endometrial neopplasia in Pten +−/− mice (AH, atypical hyperplasia, CIS, carcinoma in situ ). The source of the tissue sections is indicated. ( b ) Incidence of endometrial neoplasia in Pten +/– , Pten +/– Akt2 –/– and wild-type mice. Total number of mice examined in each group is indicated in parentheses. ( c ) BrdU incorporation in the uteri of Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice in each group are indicated in parentheses. P -values are indicated.

    Journal: Oncogene

    Article Title: The effect Akt2 deletion on tumor development in Pten+/− mice

    doi: 10.1038/onc.2011.243

    Figure Lengend Snippet: Akt2 deficiency is insufficient to inhibit the development of endometrial carcinoma in Pten +/– mice. ( a ) Histological sections representing the different grades of endometrial neopplasia in Pten +−/− mice (AH, atypical hyperplasia, CIS, carcinoma in situ ). The source of the tissue sections is indicated. ( b ) Incidence of endometrial neoplasia in Pten +/– , Pten +/– Akt2 –/– and wild-type mice. Total number of mice examined in each group is indicated in parentheses. ( c ) BrdU incorporation in the uteri of Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice in each group are indicated in parentheses. P -values are indicated.

    Article Snippet: The following primary antibodies were used: rabbit anti-pS473-Akt, (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PTEN (Biosource, Camarillo, CA, USA), mouse anti-BrdU (Dako, Carpinteria, CA, USA) and rabbit anti-cytokeratin 14 (Novocastra Laboratory, Newcastle upon Tyne, UK).

    Techniques: Mouse Assay, In Situ, BrdU Incorporation Assay

    Effect of Akt2 deficiency on tumor development in the adrenal gland and on the number of polyps in the small intestine of Pten +/– mice. ( a ) The diameter of the adrenal glands in 9-month-old female or 12-month-old male, Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice are indicated in parentheses. ( b ) Quantification of BrdU incorporation in the adrenal medulla of Pten +/– and Pten +/– Akt2 –/– mice. BrdU analysis was carried out as described in Figure 1b . ( c ) The deficiency of Akt2 did not reduce the number of polyps in the small intestine of Pten +/– mice. Quantification of the number of intestinal polyps in 9-month-old female and 12-month-old male mice. The number of polyps±s.d. per mouse is shown. The numbers of mice are indicated in parentheses (F, female; M, male).

    Journal: Oncogene

    Article Title: The effect Akt2 deletion on tumor development in Pten+/− mice

    doi: 10.1038/onc.2011.243

    Figure Lengend Snippet: Effect of Akt2 deficiency on tumor development in the adrenal gland and on the number of polyps in the small intestine of Pten +/– mice. ( a ) The diameter of the adrenal glands in 9-month-old female or 12-month-old male, Pten +/– , Pten +/– Akt2 –/– and wild-type mice. The numbers of mice are indicated in parentheses. ( b ) Quantification of BrdU incorporation in the adrenal medulla of Pten +/– and Pten +/– Akt2 –/– mice. BrdU analysis was carried out as described in Figure 1b . ( c ) The deficiency of Akt2 did not reduce the number of polyps in the small intestine of Pten +/– mice. Quantification of the number of intestinal polyps in 9-month-old female and 12-month-old male mice. The number of polyps±s.d. per mouse is shown. The numbers of mice are indicated in parentheses (F, female; M, male).

    Article Snippet: The following primary antibodies were used: rabbit anti-pS473-Akt, (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PTEN (Biosource, Camarillo, CA, USA), mouse anti-BrdU (Dako, Carpinteria, CA, USA) and rabbit anti-cytokeratin 14 (Novocastra Laboratory, Newcastle upon Tyne, UK).

    Techniques: Mouse Assay, BrdU Incorporation Assay

    Akt2 deficiency does not impair the development of high-grade PIN in Pten +/– mice. ( a ) Incidence of PIN3 and PIN4 lesions in the three prostate lobes (A, anterior, DL, dorsolateral and V, ventral) of Pten +/– and Pten +/– Akt2 –/– mice. The numbers of mice in each group are indicated. ( b ) BrdU incorporation in the prostate lobes of Pten +/– and Pten +/– Akt2 –/– mice. The numbers of mice in each group are indicated in parentheses. BrdU-positive cells were counted as described in Materials and methods. P -values were calculated for each prostate lobe in each genotype. ( c ) Quantification of K14 staining, from five mice of each genotype as described in Materials and methods.

    Journal: Oncogene

    Article Title: The effect Akt2 deletion on tumor development in Pten+/− mice

    doi: 10.1038/onc.2011.243

    Figure Lengend Snippet: Akt2 deficiency does not impair the development of high-grade PIN in Pten +/– mice. ( a ) Incidence of PIN3 and PIN4 lesions in the three prostate lobes (A, anterior, DL, dorsolateral and V, ventral) of Pten +/– and Pten +/– Akt2 –/– mice. The numbers of mice in each group are indicated. ( b ) BrdU incorporation in the prostate lobes of Pten +/– and Pten +/– Akt2 –/– mice. The numbers of mice in each group are indicated in parentheses. BrdU-positive cells were counted as described in Materials and methods. P -values were calculated for each prostate lobe in each genotype. ( c ) Quantification of K14 staining, from five mice of each genotype as described in Materials and methods.

    Article Snippet: The following primary antibodies were used: rabbit anti-pS473-Akt, (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PTEN (Biosource, Camarillo, CA, USA), mouse anti-BrdU (Dako, Carpinteria, CA, USA) and rabbit anti-cytokeratin 14 (Novocastra Laboratory, Newcastle upon Tyne, UK).

    Techniques: Mouse Assay, BrdU Incorporation Assay, Staining

    Follistatin blocks the growth arrest induced by activin A in LE6 cells. (A) LE6 cells were treated with activin A (200 ng/ml) in the presence or absence of the indicated doses of follistatin (25, 50, 100, 200, 400, 600 ng/ml). Cell proliferation was detected by CCK-8 assay. (B) LE6 cells were grown in LE media in the presence or absence of activin A (200 ng/ml), follistatin (400 ng/ml) or activin A (200 ng/ml) plus follistatin (400 ng/ml). DNA synthesis was detected by BrdU incorporation assay using FACS. (C) LE6 cells were treated with or without activin A (200 ng/ml), follistatin (400 ng/ml) or activin A (200 ng/ml) plus follistatin (400 ng/ml) for 30 min. Phosphorylated SMAD2/3 was detected by western-blot. (D) LE6 cells were treated with either media alone, activin A (200 ng/ml), follistatin (400 ng/ml) or activin(200 ng/ml) plus follistatin (400 ng/ml). Then phosphorylated Rb, cyclinD1, cyclinE, p21 WAF1/Cip1 and p15 INK4B were analyzed by western-blot.

    Journal: Cell Communication and Signaling : CCS

    Article Title: Activin A induces growth arrest through a SMAD- dependent pathway in hepatic progenitor cells

    doi: 10.1186/1478-811X-12-18

    Figure Lengend Snippet: Follistatin blocks the growth arrest induced by activin A in LE6 cells. (A) LE6 cells were treated with activin A (200 ng/ml) in the presence or absence of the indicated doses of follistatin (25, 50, 100, 200, 400, 600 ng/ml). Cell proliferation was detected by CCK-8 assay. (B) LE6 cells were grown in LE media in the presence or absence of activin A (200 ng/ml), follistatin (400 ng/ml) or activin A (200 ng/ml) plus follistatin (400 ng/ml). DNA synthesis was detected by BrdU incorporation assay using FACS. (C) LE6 cells were treated with or without activin A (200 ng/ml), follistatin (400 ng/ml) or activin A (200 ng/ml) plus follistatin (400 ng/ml) for 30 min. Phosphorylated SMAD2/3 was detected by western-blot. (D) LE6 cells were treated with either media alone, activin A (200 ng/ml), follistatin (400 ng/ml) or activin(200 ng/ml) plus follistatin (400 ng/ml). Then phosphorylated Rb, cyclinD1, cyclinE, p21 WAF1/Cip1 and p15 INK4B were analyzed by western-blot.

    Article Snippet: Then sections were incubated with rabbit anti-pan-cytokeratin antibody (1:50, DAKO, Denmark), mouse anti-activin A antibody (1:100, R & D, USA), rabbit anti-follistatin antibody (1:50, ProteinTech Group, China) or mouse anti-BrdU antibody (1:400, DAKO Denmark) at 4°C overnight.

    Techniques: CCK-8 Assay, DNA Synthesis, BrdU Incorporation Assay, FACS, Western Blot

    Follistatin accelerated oval cell proliferation in vivo . (A and D) negative control; Pan-CK positive hepatic progenitor cells ( B and C , 100 × magnification) and BrdU positive proliferating cells ( E and F , 200 × magnification) in normal sailine (NS) or follistatin treated (FST) 2-AAF/PH rats 6 day after PH were determined by immuno-histochemistry. Comparison of the number of Pan-CK positive hepatic progenitor cells (G) , BrdU positive proliferating cells (H) and Liver /body weight ratio (I) in two groups of animals. Data represent mean ± SD, n = 4–6, *P

    Journal: Cell Communication and Signaling : CCS

    Article Title: Activin A induces growth arrest through a SMAD- dependent pathway in hepatic progenitor cells

    doi: 10.1186/1478-811X-12-18

    Figure Lengend Snippet: Follistatin accelerated oval cell proliferation in vivo . (A and D) negative control; Pan-CK positive hepatic progenitor cells ( B and C , 100 × magnification) and BrdU positive proliferating cells ( E and F , 200 × magnification) in normal sailine (NS) or follistatin treated (FST) 2-AAF/PH rats 6 day after PH were determined by immuno-histochemistry. Comparison of the number of Pan-CK positive hepatic progenitor cells (G) , BrdU positive proliferating cells (H) and Liver /body weight ratio (I) in two groups of animals. Data represent mean ± SD, n = 4–6, *P

    Article Snippet: Then sections were incubated with rabbit anti-pan-cytokeratin antibody (1:50, DAKO, Denmark), mouse anti-activin A antibody (1:100, R & D, USA), rabbit anti-follistatin antibody (1:50, ProteinTech Group, China) or mouse anti-BrdU antibody (1:400, DAKO Denmark) at 4°C overnight.

    Techniques: In Vivo, Negative Control, Immunohistochemistry

    Lungs of GR Col1-Cre animals show an increase in the proliferation rate and changes in the composition of the ECM. A, Mothers were injected with BrdU 2 h before embryo collection, and proliferating cells were identified by immunohistochemistry. The proliferation index is defined as the percentage of BrdU-positive cells of the total cell number. Each bar represents three animals. B and C, Immunohistochemistry shows continuous expression of collagens I and III until E18.5 in GR Col1-Cre mutant lungs. In contrast, maturation coincides with loss of detectable collagen fibers in lungs of control littermates.

    Journal: Molecular Endocrinology

    Article Title: Glucocorticoid Activity during Lung Maturation Is Essential in Mesenchymal and Less in Alveolar Epithelial Cells

    doi: 10.1210/me.2009-0380

    Figure Lengend Snippet: Lungs of GR Col1-Cre animals show an increase in the proliferation rate and changes in the composition of the ECM. A, Mothers were injected with BrdU 2 h before embryo collection, and proliferating cells were identified by immunohistochemistry. The proliferation index is defined as the percentage of BrdU-positive cells of the total cell number. Each bar represents three animals. B and C, Immunohistochemistry shows continuous expression of collagens I and III until E18.5 in GR Col1-Cre mutant lungs. In contrast, maturation coincides with loss of detectable collagen fibers in lungs of control littermates.

    Article Snippet: Specimens were processed for immunohistochemistry as described above, and proliferating cells were detected with a monoclonal mouse anti-BrdU antibody (Bu20a; DAKO, Carpinteria, CA).

    Techniques: Injection, Immunohistochemistry, Expressing, Mutagenesis

    PCNA-expression and BrdU-incorporation. Quantitative analysis and representative images of PCNA expressing hepatocytes (A, B and C) and incorporation of BrdU (A, D and E) in animals at 24 h after pHx and daily administration of low dose of EPO (500 IU/kg bw iv; closed bars) or physiologic saline solution (open bars). Fuchsin-red (B and C) and DAB-brown (D and E) positive hepatocytes are PCNA-expressing and BrdU-incorporating cells, reflecting cells undergoing proliferation and DNA-synthesis. Note the marked reduction of these cells within the liver tissues of the EPO-treated animals (C and E). Bars represent 50 µm. Means±SEM; unpaired Student's t-test. * P

    Journal: PLoS ONE

    Article Title: Multiple Doses of Erythropoietin Impair Liver Regeneration by Increasing TNF-?, the Bax to Bcl-xL Ratio and Apoptotic Cell Death

    doi: 10.1371/journal.pone.0003924

    Figure Lengend Snippet: PCNA-expression and BrdU-incorporation. Quantitative analysis and representative images of PCNA expressing hepatocytes (A, B and C) and incorporation of BrdU (A, D and E) in animals at 24 h after pHx and daily administration of low dose of EPO (500 IU/kg bw iv; closed bars) or physiologic saline solution (open bars). Fuchsin-red (B and C) and DAB-brown (D and E) positive hepatocytes are PCNA-expressing and BrdU-incorporating cells, reflecting cells undergoing proliferation and DNA-synthesis. Note the marked reduction of these cells within the liver tissues of the EPO-treated animals (C and E). Bars represent 50 µm. Means±SEM; unpaired Student's t-test. * P

    Article Snippet: Mouse monoclonal anti-PCNA (1:100; Novocastra Laboratories, Newcastle upon Tyne, UK) and mouse monoclonal anti-BrdU (1:50; Dako Cytomation, Hamburg, Germany) were used as primary antibodies and incubated for 18 h at 4°C.

    Techniques: Expressing, BrdU Incorporation Assay, DNA Synthesis

    The receptor RAGE critically mediates the cellular response to S100A8/A9. a: Direct blockade by a specific RAGE blocking antibody reduces cellular proliferation. RAGE dependent proliferation was analyzed in normal primary, AK and SCC cells. Cells were incubated for 1 hour with a blocking anti-RAGE antibody (80μg/ml, as recommended by manufacturer) followed by S100A8/A9 stimulation for additional 24 hours. The differences in the proliferation after the blockade were assessed by BrdU incorporation (1 way Anova, Bonferroni`s Multiple test, **p

    Journal: PLoS ONE

    Article Title: S100A8/A9 Stimulates Keratinocyte Proliferation in the Development of Squamous Cell Carcinoma of the Skin via the Receptor for Advanced Glycation-End Products

    doi: 10.1371/journal.pone.0120971

    Figure Lengend Snippet: The receptor RAGE critically mediates the cellular response to S100A8/A9. a: Direct blockade by a specific RAGE blocking antibody reduces cellular proliferation. RAGE dependent proliferation was analyzed in normal primary, AK and SCC cells. Cells were incubated for 1 hour with a blocking anti-RAGE antibody (80μg/ml, as recommended by manufacturer) followed by S100A8/A9 stimulation for additional 24 hours. The differences in the proliferation after the blockade were assessed by BrdU incorporation (1 way Anova, Bonferroni`s Multiple test, **p

    Article Snippet: Double staining for RAGE and BrdU was performed using the specific RAGE antibody and specific mouse anti BrdU antibody (Millipore ).

    Techniques: Blocking Assay, Incubation, BrdU Incorporation Assay

    Endogenous S100A8/A9 is involved in cellular proliferation. a: Spontaneous secretion of S100A8/A9 from normal and SCC-derived keratinocytes. Normal and SCC-derived keratinocytes were grown in 96 well plates for 24 hours. Afterwards, supernatant was collected and preceded for the assessment of secreted S100A8/A9 using specific ELISA for S100A8/A9. b: Blockade of RAGE using anti RAGE blocking antibody reduces spontaneous proliferation of keratinocytes. Normal and SCC-derived keratinocytes were incubated with a blocking anti-RAGE antibody (8ug/100μl) for 24 hours. A decrease of the proliferation rate was detected (20–30%) based on the BrdU incorporation (t-test *p = 0.002). All the results are presented as percentage deviation from corresponding control and represent the mean +/- SD of duplicate values.

    Journal: PLoS ONE

    Article Title: S100A8/A9 Stimulates Keratinocyte Proliferation in the Development of Squamous Cell Carcinoma of the Skin via the Receptor for Advanced Glycation-End Products

    doi: 10.1371/journal.pone.0120971

    Figure Lengend Snippet: Endogenous S100A8/A9 is involved in cellular proliferation. a: Spontaneous secretion of S100A8/A9 from normal and SCC-derived keratinocytes. Normal and SCC-derived keratinocytes were grown in 96 well plates for 24 hours. Afterwards, supernatant was collected and preceded for the assessment of secreted S100A8/A9 using specific ELISA for S100A8/A9. b: Blockade of RAGE using anti RAGE blocking antibody reduces spontaneous proliferation of keratinocytes. Normal and SCC-derived keratinocytes were incubated with a blocking anti-RAGE antibody (8ug/100μl) for 24 hours. A decrease of the proliferation rate was detected (20–30%) based on the BrdU incorporation (t-test *p = 0.002). All the results are presented as percentage deviation from corresponding control and represent the mean +/- SD of duplicate values.

    Article Snippet: Double staining for RAGE and BrdU was performed using the specific RAGE antibody and specific mouse anti BrdU antibody (Millipore ).

    Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Incubation, BrdU Incorporation Assay, T-Test

    Effect of L-serine treatment on endogenous NSC proliferation in the IBZ of cortex 6 days after pMCAO. (A) Representative examples of BrdU- and nestin-positive cells: a, sham-operated group; b, vehicle group; c, L-serine (168 mg/kg) treated group; d, L-serine (168 mg/kg) and D-cycloserine (15 mg/kg) treated group. (B) Mean values of the number of BrdU + /nestin + NSCs in the IBZ of cortex (n = 4–6). * p

    Journal: PLoS ONE

    Article Title: L-Serine Treatment May Improve Neurorestoration of Rats after Permanent Focal Cerebral Ischemia Potentially Through Improvement of Neurorepair

    doi: 10.1371/journal.pone.0093405

    Figure Lengend Snippet: Effect of L-serine treatment on endogenous NSC proliferation in the IBZ of cortex 6 days after pMCAO. (A) Representative examples of BrdU- and nestin-positive cells: a, sham-operated group; b, vehicle group; c, L-serine (168 mg/kg) treated group; d, L-serine (168 mg/kg) and D-cycloserine (15 mg/kg) treated group. (B) Mean values of the number of BrdU + /nestin + NSCs in the IBZ of cortex (n = 4–6). * p

    Article Snippet: Chemicals L-Serine, 2,3,5-triphenyltetrazolium chloride (TTC), 5-bromo-2′-deoxyuridine (B5002, BrdU), D-cycloserine (C3909), mouse monoclonal anti-BrdU antibody (B2513), rabbit anti-nestin antibody (N5413), fluorescein (FITC)-conjugated goat anti-rabbit IgG (whole molecule) (F9887) were purchased from Sigma-Aldrich Corporation (Saint Louis, USA).

    Techniques:

    Temporal order of V0 excitatory neurogenesis. In all the experiments, fish in which a small number of V0 excitatory neurons expressed GFP were obtained by injecting the vglut2a :lDl-GFP DNA construct into Tg[ dbx1b :Cre] transgenic fish. A , Examples of three different time points. Top, 1.5 dpf. Two V0-eA neurons are labeled. Middle, 2.5 dpf. A V0-eB neuron is labeled (arrows indicate growing bifurcating axon). Bottom, A V0-eD neuron is labeled. B , Observation frequency of three classes of V0 excitatory neurons at three different time points. C , Schematics of BrdU incorporation experiments. BrdU was applied at a given time point, and embryos were incubated in the presence of BrdU until 72 hpf. Then, animals were moved to BrdU-free solution. At 120 hpf (5 dpf), neuronal morphology was examined. The animals were then processed for BrdU immunohistochemistry. D , Examples of V0-eB neurons. In each panel, the top images shows a stack of images from the living fish at 5 dpf, while the bottom three show images after BrdU immunohistochemistry. The GFP images are in green and the BrdU images are in red. Left, BrdU was applied at 24 hpf in this animal. The cell is positive for BrdU. Right, BrdU was applied at 36 hpf in this animal. The cell is negative for BrdU. E , Summary of BrdU incorporation experiments. Percentage of BrdU-positive and -negative cells for each cell type at a given time point of BrdU application is indicated. Scale bar, 20 μm.

    Journal: The Journal of Neuroscience

    Article Title: Generation of Multiple Classes of V0 Neurons in Zebrafish Spinal Cord: Progenitor Heterogeneity and Temporal Control of Neuronal Diversity

    doi: 10.1523/JNEUROSCI.5500-11.2012

    Figure Lengend Snippet: Temporal order of V0 excitatory neurogenesis. In all the experiments, fish in which a small number of V0 excitatory neurons expressed GFP were obtained by injecting the vglut2a :lDl-GFP DNA construct into Tg[ dbx1b :Cre] transgenic fish. A , Examples of three different time points. Top, 1.5 dpf. Two V0-eA neurons are labeled. Middle, 2.5 dpf. A V0-eB neuron is labeled (arrows indicate growing bifurcating axon). Bottom, A V0-eD neuron is labeled. B , Observation frequency of three classes of V0 excitatory neurons at three different time points. C , Schematics of BrdU incorporation experiments. BrdU was applied at a given time point, and embryos were incubated in the presence of BrdU until 72 hpf. Then, animals were moved to BrdU-free solution. At 120 hpf (5 dpf), neuronal morphology was examined. The animals were then processed for BrdU immunohistochemistry. D , Examples of V0-eB neurons. In each panel, the top images shows a stack of images from the living fish at 5 dpf, while the bottom three show images after BrdU immunohistochemistry. The GFP images are in green and the BrdU images are in red. Left, BrdU was applied at 24 hpf in this animal. The cell is positive for BrdU. Right, BrdU was applied at 36 hpf in this animal. The cell is negative for BrdU. E , Summary of BrdU incorporation experiments. Percentage of BrdU-positive and -negative cells for each cell type at a given time point of BrdU application is indicated. Scale bar, 20 μm.

    Article Snippet: We used the following primary antibodies: anti-GAD65/67 antibody (GC3108, Sigma, 1:500), rabbit anti-Pax2 (ab37129, Abcam, 1:200), rabbit anti-GFP ( , Invitrogen, 1:200), rat anti-GFP (04404-26, Nacalai, 1:200), mouse anti-BrdU (G3G4, Developmental Studies Hybridoma Bank, 1:200), rabbit anti-Evx2, and guinea pig anti-Evx2 (these anti-Evx2 were generated in our laboratory using bacterially expressed proteins).

    Techniques: Fluorescence In Situ Hybridization, Construct, Transgenic Assay, Labeling, BrdU Incorporation Assay, Incubation, Immunohistochemistry

    Neurogenesis and angiogenesis effects of human mesenchymal stem cells (hMSCs) in a stroke rat model. (A) Representative images of immunofluorescence staining with anti-BrdU (red)/anti-doublecortin (DCX; green) for analysis of neurogenesis. (B) Quantitative analysis of neurogenesis expressed as the proliferating cell number with anti-BrdU/anti-DCX positive cells in the subventricular zone. (C) Representative images of immunofluorescence staining with anti-vWF (red)/4’,6-diamidino-2-phenylindole (blue) for analysis of angiogenesis. (D) Quantitative analysis of angiogenesis expressed as the anti–von Willebrand factor positive area in the striatum. The quantitation of stained cells was performed in 6 fields per section. The data are presented as mean + SD (* P

    Journal: Cell Transplantation

    Article Title: Serum-mediated Activation of Bone Marrow–derived Mesenchymal Stem Cells in Ischemic Stroke Patients

    doi: 10.1177/0963689718755404

    Figure Lengend Snippet: Neurogenesis and angiogenesis effects of human mesenchymal stem cells (hMSCs) in a stroke rat model. (A) Representative images of immunofluorescence staining with anti-BrdU (red)/anti-doublecortin (DCX; green) for analysis of neurogenesis. (B) Quantitative analysis of neurogenesis expressed as the proliferating cell number with anti-BrdU/anti-DCX positive cells in the subventricular zone. (C) Representative images of immunofluorescence staining with anti-vWF (red)/4’,6-diamidino-2-phenylindole (blue) for analysis of angiogenesis. (D) Quantitative analysis of angiogenesis expressed as the anti–von Willebrand factor positive area in the striatum. The quantitation of stained cells was performed in 6 fields per section. The data are presented as mean + SD (* P

    Article Snippet: Neurogenesis and angiogenesis effects were calculated using immunostaining with mouse anti-BrdU (diluted 1:50, Abcam, Cambridge, United Kingdom)/rabbit anti-doublecortin (anti-DCX, diluted 1:200, Abcam) or rabbit anti–von Willebrand factor (anti-vWF, diluted 1:200, Chemicon, Temecula, CA, USA).

    Techniques: Immunofluorescence, Staining, Quantitation Assay

    Premature postmitotic differentiation and depletion of proliferative neural precursor cells in the ventricular zone. A–R , Immunohistochemical analysis of SOX2 and HUC/D ( A–I ), SOX3 and HUC/D ( J–L ), and BrdU incorporation ( P–R ), as well as Hes5 nonradioactive in situ hybridization ( M–O ) on coronal midbrain sections of wild-type (WT), Fgfr1 cko ;Fgfr2 cko ( R1cko;R2cko ), and Fgfr1 cko ;Fgfr2 cko ;Fgfr3 null ( R1cko;R2cko;R3null ) embryos at E9.5–E11.5. S , The thickness of the ventricular zone (SOX2-positive) and the marginal zone (HUC/D-positive) was quantified (mean ± SD) at E11.5. G , The position at which layer thickness was measured is shown with white lines. For quantification of BrdU incorporation, the ventricular zone was divided into an approximately two-cell-layer-thick periventricular zone (zone1) and basal zone (zone2), visualized with broken lines ( P–R ). T , The proportion (mean ± SD) of BrdU-positive nuclei in zone1, zone2, and in the entire ventricular zone (total). White arrows point to decreased SOX2-positive layer and increased HUC/D-positive layer ( C , E , F , H , I ), downregulated SOX3 expression ( K , L ), and periventricular BrdU-positive cells ( Q , R ) in the mutants. Z1, Zone1; Z2, zone2. Asterisks indicate Hes5 -negative ventral domain. DAPI, 4′,6′-Diamidino-2-phenylindole. Scale bars, 100 μm. * p

    Journal: The Journal of Neuroscience

    Article Title: Fibroblast Growth Factor Receptors Cooperate to Regulate Neural Progenitor Properties in the Developing Midbrain and Hindbrain

    doi: 10.1523/JNEUROSCI.0192-07.2007

    Figure Lengend Snippet: Premature postmitotic differentiation and depletion of proliferative neural precursor cells in the ventricular zone. A–R , Immunohistochemical analysis of SOX2 and HUC/D ( A–I ), SOX3 and HUC/D ( J–L ), and BrdU incorporation ( P–R ), as well as Hes5 nonradioactive in situ hybridization ( M–O ) on coronal midbrain sections of wild-type (WT), Fgfr1 cko ;Fgfr2 cko ( R1cko;R2cko ), and Fgfr1 cko ;Fgfr2 cko ;Fgfr3 null ( R1cko;R2cko;R3null ) embryos at E9.5–E11.5. S , The thickness of the ventricular zone (SOX2-positive) and the marginal zone (HUC/D-positive) was quantified (mean ± SD) at E11.5. G , The position at which layer thickness was measured is shown with white lines. For quantification of BrdU incorporation, the ventricular zone was divided into an approximately two-cell-layer-thick periventricular zone (zone1) and basal zone (zone2), visualized with broken lines ( P–R ). T , The proportion (mean ± SD) of BrdU-positive nuclei in zone1, zone2, and in the entire ventricular zone (total). White arrows point to decreased SOX2-positive layer and increased HUC/D-positive layer ( C , E , F , H , I ), downregulated SOX3 expression ( K , L ), and periventricular BrdU-positive cells ( Q , R ) in the mutants. Z1, Zone1; Z2, zone2. Asterisks indicate Hes5 -negative ventral domain. DAPI, 4′,6′-Diamidino-2-phenylindole. Scale bars, 100 μm. * p

    Article Snippet: Antibodies used for immunohistochemistry were rabbit anti-ALDH1 (1:500; Abcam, Cambridge, UK), mouse anti-BrdU (1:400; GE Healthcare), mouse anti-HuC/D (1:500; Invitrogen, Eugene, OR), rabbit anti-LMX1a (1:300; from Michael German, University of California at San Francisco, San Francisco, CA), rabbit anti-SOX2 (1:500; Millipore), rabbit anti-SOX3 (1:500; from Thomas Edlund, Umeå University, Umeå, Sweden), and rabbit and mouse anti-tyrosine hydroxylase (TH; 1:500; Millipore).

    Techniques: Immunohistochemistry, BrdU Incorporation Assay, In Situ Hybridization, Expressing

    Scatter plots display the correlations between the variables related with the hippocampal neuroplasticity and the hippocampal-dependent memory behavioral tests. Note: In the vmPFC HFS animals, the Morris water-maze target quadrant is positively correlated with the Dcx cell count ( A ), while the novel-object recognition task with no-HFS prior to testing shows positive correlation with the Dcx cell count ( C ) as well as the NeuN/Rbfox3 gene expression ( D ). No correlational association was found between the Morris water-maze and BrdU cell count after chronic vmPFC high-frequency stimulation ( B ). DOI: http://dx.doi.org/10.7554/eLife.04803.009

    Journal: eLife

    Article Title: Ventromedial prefrontal cortex stimulation enhances memory and hippocampal neurogenesis in the middle-aged rats

    doi: 10.7554/eLife.04803

    Figure Lengend Snippet: Scatter plots display the correlations between the variables related with the hippocampal neuroplasticity and the hippocampal-dependent memory behavioral tests. Note: In the vmPFC HFS animals, the Morris water-maze target quadrant is positively correlated with the Dcx cell count ( A ), while the novel-object recognition task with no-HFS prior to testing shows positive correlation with the Dcx cell count ( C ) as well as the NeuN/Rbfox3 gene expression ( D ). No correlational association was found between the Morris water-maze and BrdU cell count after chronic vmPFC high-frequency stimulation ( B ). DOI: http://dx.doi.org/10.7554/eLife.04803.009

    Article Snippet: After pre-blocking for 30 min in PBS-Triton (PBS-T) with 10% normal donkey serum, two double labeling stainings were carried out on the vmPFC HFS hippocampal sections using goat anti-Dcx (1:50) and rabbit anti-c-Fos antibody (1:500); as well as mouse anti-BrdU (1:50) and goat anti-c-Fos (1:100) as primary antibodies (all antibodies, Santa Cruz Biotechnology, Inc.) in 5% normal donkey serum for 3-day incubation.

    Techniques: Cell Counting, Expressing

    Effects of chronic vmPFC HFS on the hippocampal neuronal activity by c-Fos-ir ( A – B ) and the morphological changes related to neurogenesis functions ( E – I ). Note: VmPFC HFS increased the number of c-Fos-ir positive cells in the subiculum, DG, and a marginal difference (p = 0.059) in the CA1 field of the hippocampus as compared to the sham ( A–B ; scale bars: 500 μm, low-power magnification; 250 μm, high-power magnification). In neurogenesis-related morphology, after chronic vmPFC HFS, there was an increase of surviving BrdU-positive cells ( E–F , scale bar: 500 μm), and neural progenitors—Dcx-positive cells ( G–H ; scale bars: 300 μm, low-power magnification; 50 μm, high-power magnification). For correlational analysis, there was a strong relationship between the BrdU and Dcx cell-count ( I ). Importantly, the neurogenic zone of the DG was also highly correlated with the SUB and CA1 field of the hippocampus ( C–D ), indicating that these regions were functionally associated with memory functions after chronic vmPFC HFS. Abbreviations: SUB, subiculum; DG, dentate gyrus; CA1, CA1 field of the hippocampus; CA3, CA3 field of the hippocampus; vmPFC HFS, high-frequency stimulation of the ventromedial prefrontal cortex; BrdU, 5-bromo-2′-deoxyuridine; and Dcx, doublecourtin. Indication: *, significant difference from the sham rats, (p

    Journal: eLife

    Article Title: Ventromedial prefrontal cortex stimulation enhances memory and hippocampal neurogenesis in the middle-aged rats

    doi: 10.7554/eLife.04803

    Figure Lengend Snippet: Effects of chronic vmPFC HFS on the hippocampal neuronal activity by c-Fos-ir ( A – B ) and the morphological changes related to neurogenesis functions ( E – I ). Note: VmPFC HFS increased the number of c-Fos-ir positive cells in the subiculum, DG, and a marginal difference (p = 0.059) in the CA1 field of the hippocampus as compared to the sham ( A–B ; scale bars: 500 μm, low-power magnification; 250 μm, high-power magnification). In neurogenesis-related morphology, after chronic vmPFC HFS, there was an increase of surviving BrdU-positive cells ( E–F , scale bar: 500 μm), and neural progenitors—Dcx-positive cells ( G–H ; scale bars: 300 μm, low-power magnification; 50 μm, high-power magnification). For correlational analysis, there was a strong relationship between the BrdU and Dcx cell-count ( I ). Importantly, the neurogenic zone of the DG was also highly correlated with the SUB and CA1 field of the hippocampus ( C–D ), indicating that these regions were functionally associated with memory functions after chronic vmPFC HFS. Abbreviations: SUB, subiculum; DG, dentate gyrus; CA1, CA1 field of the hippocampus; CA3, CA3 field of the hippocampus; vmPFC HFS, high-frequency stimulation of the ventromedial prefrontal cortex; BrdU, 5-bromo-2′-deoxyuridine; and Dcx, doublecourtin. Indication: *, significant difference from the sham rats, (p

    Article Snippet: After pre-blocking for 30 min in PBS-Triton (PBS-T) with 10% normal donkey serum, two double labeling stainings were carried out on the vmPFC HFS hippocampal sections using goat anti-Dcx (1:50) and rabbit anti-c-Fos antibody (1:500); as well as mouse anti-BrdU (1:50) and goat anti-c-Fos (1:100) as primary antibodies (all antibodies, Santa Cruz Biotechnology, Inc.) in 5% normal donkey serum for 3-day incubation.

    Techniques: Activity Assay, Cell Counting

    Effects of chronic vmPFC HFS on the Golgi measurement of dendritic spine density and immunofluorescence labeling of neurogenesis-related cell function in the hippocampal DG area. Note: there was an increase in the secondary, but not the primary dendritic spine density of the Golgi-impregnated cells in the DG area of the hippocampus ( A–B ; scale bar: 10 μm). Representative confocal images ( C ) are demonstrated for the localization of Dcx (green, C-i ), c-Fos (red, C-ii ; or green, C-v ), and BrdU-labeled (red, C-iv ) immunofluorescence positive cells. Merged images showed the co-localization of Dcx and c-Fos ( C-iii ), as well as BrdU and c-Fos ( C-vi ) in the hippocampal DG region (scale bar: 40 μm). Indication: *, significant difference from the sham rats, (p

    Journal: eLife

    Article Title: Ventromedial prefrontal cortex stimulation enhances memory and hippocampal neurogenesis in the middle-aged rats

    doi: 10.7554/eLife.04803

    Figure Lengend Snippet: Effects of chronic vmPFC HFS on the Golgi measurement of dendritic spine density and immunofluorescence labeling of neurogenesis-related cell function in the hippocampal DG area. Note: there was an increase in the secondary, but not the primary dendritic spine density of the Golgi-impregnated cells in the DG area of the hippocampus ( A–B ; scale bar: 10 μm). Representative confocal images ( C ) are demonstrated for the localization of Dcx (green, C-i ), c-Fos (red, C-ii ; or green, C-v ), and BrdU-labeled (red, C-iv ) immunofluorescence positive cells. Merged images showed the co-localization of Dcx and c-Fos ( C-iii ), as well as BrdU and c-Fos ( C-vi ) in the hippocampal DG region (scale bar: 40 μm). Indication: *, significant difference from the sham rats, (p

    Article Snippet: After pre-blocking for 30 min in PBS-Triton (PBS-T) with 10% normal donkey serum, two double labeling stainings were carried out on the vmPFC HFS hippocampal sections using goat anti-Dcx (1:50) and rabbit anti-c-Fos antibody (1:500); as well as mouse anti-BrdU (1:50) and goat anti-c-Fos (1:100) as primary antibodies (all antibodies, Santa Cruz Biotechnology, Inc.) in 5% normal donkey serum for 3-day incubation.

    Techniques: Immunofluorescence, Labeling, Cell Function Assay