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  • 99
    Becton Dickinson mouse monoclonal anti brdu
    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 <t>BrdU</t> 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, <t>CRALBP,</t> 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
    Mouse Monoclonal Anti Brdu, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse monoclonal anti brdu primary antibody
    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 <t>BrdU</t> 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, <t>CRALBP,</t> 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
    Mouse Monoclonal Anti Brdu Primary Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 81/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson monoclonal anti brdu antibody
    Spry1-2 −/− Shows Increased Proliferation, Precocious Progenitor Cell Maturation and Neurogenesis at E12.5 (A-C') Immunofluorescence analysis for Blbp (A-B', red), and Nestin (A-A' and C-C', green) in the WT and Spry1-2 −/− brains. Blbp, a marker for radial glia, is strongly up-regulated in the dorsal/medial pallium (arrowheads in B'). (D-D') Short-pulse <t>BrdU</t> labeling of WT (D) and Spry1-2 −/− (D'). BrdU was injected to pregnant females and E12.5 embryos were collected after 30', and coronal sections were stained with anti-BrdU antibody. More BrdU + cells are present in the dorsal pallium in Spry1-2 −/− cortex (D') than in WT (D). (E-E') RNA in situ hybridization for Tbr2 in WT and Spry1-2 −/− brains, showing the increase of Tbr2 staining in preplate (red arrowhead) and in VZ/SVZ region (black arrowhead). (F-F') RNA in situ hybridization for Etv1 in WT and Spry1-2 −/− brains, showing the increase of Etv1 staining in the preplate (arrowheads). (G-H') <t>β-tubulin</t> (green) and Blbp (red) immunodetection in WT and Spry1-2 −/− brains. Arrowheads in H' point to increase preplate thickness in Spry1-2 −/− null mice, in a region of high Blbp expression (H'). Scale bars: (A-C'), (F-G'), (H-I') 200 μm, (D-D'), 250 μm.
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    Becton Dickinson mouse monoclonal anti brdu antibody beckton dickinson
    Spry1-2 −/− Shows Increased Proliferation, Precocious Progenitor Cell Maturation and Neurogenesis at E12.5 (A-C') Immunofluorescence analysis for Blbp (A-B', red), and Nestin (A-A' and C-C', green) in the WT and Spry1-2 −/− brains. Blbp, a marker for radial glia, is strongly up-regulated in the dorsal/medial pallium (arrowheads in B'). (D-D') Short-pulse <t>BrdU</t> labeling of WT (D) and Spry1-2 −/− (D'). BrdU was injected to pregnant females and E12.5 embryos were collected after 30', and coronal sections were stained with anti-BrdU antibody. More BrdU + cells are present in the dorsal pallium in Spry1-2 −/− cortex (D') than in WT (D). (E-E') RNA in situ hybridization for Tbr2 in WT and Spry1-2 −/− brains, showing the increase of Tbr2 staining in preplate (red arrowhead) and in VZ/SVZ region (black arrowhead). (F-F') RNA in situ hybridization for Etv1 in WT and Spry1-2 −/− brains, showing the increase of Etv1 staining in the preplate (arrowheads). (G-H') <t>β-tubulin</t> (green) and Blbp (red) immunodetection in WT and Spry1-2 −/− brains. Arrowheads in H' point to increase preplate thickness in Spry1-2 −/− null mice, in a region of high Blbp expression (H'). Scale bars: (A-C'), (F-G'), (H-I') 200 μm, (D-D'), 250 μm.
    Mouse Monoclonal Anti Brdu Antibody Beckton Dickinson, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse monoclonal anti brdu fitc antibody
    Spry1-2 −/− Shows Increased Proliferation, Precocious Progenitor Cell Maturation and Neurogenesis at E12.5 (A-C') Immunofluorescence analysis for Blbp (A-B', red), and Nestin (A-A' and C-C', green) in the WT and Spry1-2 −/− brains. Blbp, a marker for radial glia, is strongly up-regulated in the dorsal/medial pallium (arrowheads in B'). (D-D') Short-pulse <t>BrdU</t> labeling of WT (D) and Spry1-2 −/− (D'). BrdU was injected to pregnant females and E12.5 embryos were collected after 30', and coronal sections were stained with anti-BrdU antibody. More BrdU + cells are present in the dorsal pallium in Spry1-2 −/− cortex (D') than in WT (D). (E-E') RNA in situ hybridization for Tbr2 in WT and Spry1-2 −/− brains, showing the increase of Tbr2 staining in preplate (red arrowhead) and in VZ/SVZ region (black arrowhead). (F-F') RNA in situ hybridization for Etv1 in WT and Spry1-2 −/− brains, showing the increase of Etv1 staining in the preplate (arrowheads). (G-H') <t>β-tubulin</t> (green) and Blbp (red) immunodetection in WT and Spry1-2 −/− brains. Arrowheads in H' point to increase preplate thickness in Spry1-2 −/− null mice, in a region of high Blbp expression (H'). Scale bars: (A-C'), (F-G'), (H-I') 200 μm, (D-D'), 250 μm.
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    Becton Dickinson fitc conjugated mouse monoclonal anti brdu antibody
    Spry1-2 −/− Shows Increased Proliferation, Precocious Progenitor Cell Maturation and Neurogenesis at E12.5 (A-C') Immunofluorescence analysis for Blbp (A-B', red), and Nestin (A-A' and C-C', green) in the WT and Spry1-2 −/− brains. Blbp, a marker for radial glia, is strongly up-regulated in the dorsal/medial pallium (arrowheads in B'). (D-D') Short-pulse <t>BrdU</t> labeling of WT (D) and Spry1-2 −/− (D'). BrdU was injected to pregnant females and E12.5 embryos were collected after 30', and coronal sections were stained with anti-BrdU antibody. More BrdU + cells are present in the dorsal pallium in Spry1-2 −/− cortex (D') than in WT (D). (E-E') RNA in situ hybridization for Tbr2 in WT and Spry1-2 −/− brains, showing the increase of Tbr2 staining in preplate (red arrowhead) and in VZ/SVZ region (black arrowhead). (F-F') RNA in situ hybridization for Etv1 in WT and Spry1-2 −/− brains, showing the increase of Etv1 staining in the preplate (arrowheads). (G-H') <t>β-tubulin</t> (green) and Blbp (red) immunodetection in WT and Spry1-2 −/− brains. Arrowheads in H' point to increase preplate thickness in Spry1-2 −/− null mice, in a region of high Blbp expression (H'). Scale bars: (A-C'), (F-G'), (H-I') 200 μm, (D-D'), 250 μm.
    Fitc Conjugated Mouse Monoclonal Anti Brdu Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson fitc labeled mouse monoclonal anti brdu antibody
    Spry1-2 −/− Shows Increased Proliferation, Precocious Progenitor Cell Maturation and Neurogenesis at E12.5 (A-C') Immunofluorescence analysis for Blbp (A-B', red), and Nestin (A-A' and C-C', green) in the WT and Spry1-2 −/− brains. Blbp, a marker for radial glia, is strongly up-regulated in the dorsal/medial pallium (arrowheads in B'). (D-D') Short-pulse <t>BrdU</t> labeling of WT (D) and Spry1-2 −/− (D'). BrdU was injected to pregnant females and E12.5 embryos were collected after 30', and coronal sections were stained with anti-BrdU antibody. More BrdU + cells are present in the dorsal pallium in Spry1-2 −/− cortex (D') than in WT (D). (E-E') RNA in situ hybridization for Tbr2 in WT and Spry1-2 −/− brains, showing the increase of Tbr2 staining in preplate (red arrowhead) and in VZ/SVZ region (black arrowhead). (F-F') RNA in situ hybridization for Etv1 in WT and Spry1-2 −/− brains, showing the increase of Etv1 staining in the preplate (arrowheads). (G-H') <t>β-tubulin</t> (green) and Blbp (red) immunodetection in WT and Spry1-2 −/− brains. Arrowheads in H' point to increase preplate thickness in Spry1-2 −/− null mice, in a region of high Blbp expression (H'). Scale bars: (A-C'), (F-G'), (H-I') 200 μm, (D-D'), 250 μm.
    Fitc Labeled Mouse Monoclonal Anti Brdu Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson rat monoclonal anti brdu
    Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and <t>CldU</t> (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with <t>BrdU</t> at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p
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    Becton Dickinson mouse monoclonal antibromodeoxyuridine anti brdu
    Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and <t>CldU</t> (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with <t>BrdU</t> at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p
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    Becton Dickinson mouse anti brdu dna monoclonal antibody
    Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and <t>CldU</t> (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with <t>BrdU</t> at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p
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    Becton Dickinson biotinylated brdu mouse monoclonal
    Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and <t>CldU</t> (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with <t>BrdU</t> at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p
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    Becton Dickinson mouse anti brdu monoclonal anitbody
    Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and <t>CldU</t> (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with <t>BrdU</t> at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p
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    Becton Dickinson mouse anti brdu monoclonal antibody b44
    Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and <t>CldU</t> (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with <t>BrdU</t> at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p
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    Becton Dickinson anti brdu mouse monoclonal igg1
    Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and <t>CldU</t> (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with <t>BrdU</t> at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p
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    Becton Dickinson anti brdu monoclonal antibodies mabs
    Proliferation of CD4 + FoxP3 + Treg population after ablative conditioning . B6 mice were subjected to 9.5 Gy TBI. On the following day, mice received 5 × 10 6 T-depleted BM cells isolated from Thy1.1 congenic B6 mice. Mice were killed 20, 35, and 56 days post-BMT and spleen and lymph nodes subjected to FAC analysis. Four days before death, mice were placed on <t>BrdU</t> (0.8 mg/mL) drinking water. (A) Representative dot plot showing the Thy1.1 (donor BM) staining and IgG of BrdU staining of the gated CD4 + Foxp3 + cells in the spleen (left panels) and lymph nodes (right panels). (B) Percentage BrdU + cells in the host (Thy1.1 − ) and donor (Thy1.1 + ) CD4 + Foxp3 + and CD4 + Foxp3 − compartments in the spleen (left) and lymph nodes (right). (C) Wild-type B6 mice (n = 3) were placed on BrdU drinking water for 4 days, killed, and the spleen and lymph nodes subjected to FACS analysis. Representative dot plot showing CD4 and Foxp3 expression with corresponding histograms illustrating staining by isotype control (IgG) and anti-BrdU <t>mAb</t> of the gated CD4 + FoxP3 + and CD4 + Foxp3 − populations in the spleen (left) and lymph nodes (right). Data in panel B are mean plus or minus SEM for 3 mice per group per time point.
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    Becton Dickinson fluorescein isothiocyanate conjugated mouse anti brdu monoclonal antibody
    Proliferation of CD4 + FoxP3 + Treg population after ablative conditioning . B6 mice were subjected to 9.5 Gy TBI. On the following day, mice received 5 × 10 6 T-depleted BM cells isolated from Thy1.1 congenic B6 mice. Mice were killed 20, 35, and 56 days post-BMT and spleen and lymph nodes subjected to FAC analysis. Four days before death, mice were placed on <t>BrdU</t> (0.8 mg/mL) drinking water. (A) Representative dot plot showing the Thy1.1 (donor BM) staining and IgG of BrdU staining of the gated CD4 + Foxp3 + cells in the spleen (left panels) and lymph nodes (right panels). (B) Percentage BrdU + cells in the host (Thy1.1 − ) and donor (Thy1.1 + ) CD4 + Foxp3 + and CD4 + Foxp3 − compartments in the spleen (left) and lymph nodes (right). (C) Wild-type B6 mice (n = 3) were placed on BrdU drinking water for 4 days, killed, and the spleen and lymph nodes subjected to FACS analysis. Representative dot plot showing CD4 and Foxp3 expression with corresponding histograms illustrating staining by isotype control (IgG) and anti-BrdU <t>mAb</t> of the gated CD4 + FoxP3 + and CD4 + Foxp3 − populations in the spleen (left) and lymph nodes (right). Data in panel B are mean plus or minus SEM for 3 mice per group per time point.
    Fluorescein Isothiocyanate Conjugated Mouse Anti Brdu Monoclonal Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson biotinylated mouse monoclonal anti 5 bromo 2 deoxyuridine brdu
    Proliferation of CD4 + FoxP3 + Treg population after ablative conditioning . B6 mice were subjected to 9.5 Gy TBI. On the following day, mice received 5 × 10 6 T-depleted BM cells isolated from Thy1.1 congenic B6 mice. Mice were killed 20, 35, and 56 days post-BMT and spleen and lymph nodes subjected to FAC analysis. Four days before death, mice were placed on <t>BrdU</t> (0.8 mg/mL) drinking water. (A) Representative dot plot showing the Thy1.1 (donor BM) staining and IgG of BrdU staining of the gated CD4 + Foxp3 + cells in the spleen (left panels) and lymph nodes (right panels). (B) Percentage BrdU + cells in the host (Thy1.1 − ) and donor (Thy1.1 + ) CD4 + Foxp3 + and CD4 + Foxp3 − compartments in the spleen (left) and lymph nodes (right). (C) Wild-type B6 mice (n = 3) were placed on BrdU drinking water for 4 days, killed, and the spleen and lymph nodes subjected to FACS analysis. Representative dot plot showing CD4 and Foxp3 expression with corresponding histograms illustrating staining by isotype control (IgG) and anti-BrdU <t>mAb</t> of the gated CD4 + FoxP3 + and CD4 + Foxp3 − populations in the spleen (left) and lymph nodes (right). Data in panel B are mean plus or minus SEM for 3 mice per group per time point.
    Biotinylated Mouse Monoclonal Anti 5 Bromo 2 Deoxyuridine Brdu, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proliferation of CD4 + FoxP3 + Treg population after ablative conditioning . B6 mice were subjected to 9.5 Gy TBI. On the following day, mice received 5 × 10 6 T-depleted BM cells isolated from Thy1.1 congenic B6 mice. Mice were killed 20, 35, and 56 days post-BMT and spleen and lymph nodes subjected to FAC analysis. Four days before death, mice were placed on <t>BrdU</t> (0.8 mg/mL) drinking water. (A) Representative dot plot showing the Thy1.1 (donor BM) staining and IgG of BrdU staining of the gated CD4 + Foxp3 + cells in the spleen (left panels) and lymph nodes (right panels). (B) Percentage BrdU + cells in the host (Thy1.1 − ) and donor (Thy1.1 + ) CD4 + Foxp3 + and CD4 + Foxp3 − compartments in the spleen (left) and lymph nodes (right). (C) Wild-type B6 mice (n = 3) were placed on BrdU drinking water for 4 days, killed, and the spleen and lymph nodes subjected to FACS analysis. Representative dot plot showing CD4 and Foxp3 expression with corresponding histograms illustrating staining by isotype control (IgG) and anti-BrdU <t>mAb</t> of the gated CD4 + FoxP3 + and CD4 + Foxp3 − populations in the spleen (left) and lymph nodes (right). Data in panel B are mean plus or minus SEM for 3 mice per group per time point.
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    Proliferation of CD4 + FoxP3 + Treg population after ablative conditioning . B6 mice were subjected to 9.5 Gy TBI. On the following day, mice received 5 × 10 6 T-depleted BM cells isolated from Thy1.1 congenic B6 mice. Mice were killed 20, 35, and 56 days post-BMT and spleen and lymph nodes subjected to FAC analysis. Four days before death, mice were placed on <t>BrdU</t> (0.8 mg/mL) drinking water. (A) Representative dot plot showing the Thy1.1 (donor BM) staining and IgG of BrdU staining of the gated CD4 + Foxp3 + cells in the spleen (left panels) and lymph nodes (right panels). (B) Percentage BrdU + cells in the host (Thy1.1 − ) and donor (Thy1.1 + ) CD4 + Foxp3 + and CD4 + Foxp3 − compartments in the spleen (left) and lymph nodes (right). (C) Wild-type B6 mice (n = 3) were placed on BrdU drinking water for 4 days, killed, and the spleen and lymph nodes subjected to FACS analysis. Representative dot plot showing CD4 and Foxp3 expression with corresponding histograms illustrating staining by isotype control (IgG) and anti-BrdU <t>mAb</t> of the gated CD4 + FoxP3 + and CD4 + Foxp3 − populations in the spleen (left) and lymph nodes (right). Data in panel B are mean plus or minus SEM for 3 mice per group per time point.
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    Becton Dickinson anti brdu fluorescein conjugated mouse monoclonal antibody
    H4K16Ac levels peak at S phase and drop dramatically in the G 2 /M transition. ( A ) Mammalian fibroblasts were pulsed with <t>BrdU,</t> stained with H4K16Ac antibodies as indicated, and analyzed by immunofluorescence for colocalization of BrdU and H4K16Ac. Two different samples are shown (Exp1 and Exp2). ( B ) Same cells as in A were pulsed with BrdU, incubated with H4K16Ac antibody and 7AAD (DNA content marker), and analyzed by FACS for the levels of H4K16Ac at each stage of the cell cycle. ( Upper ). Distribution of H4K16Ac levels in cells at each stage of the cell cycle is shown graphically and numerically, with 100% representing the levels of H4K16Ac in G 1 phase. ( C ) Immunofluorescence as in A, but in this case with the mitotic marker H3S28P and H4K16Ac.
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    Becton Dickinson mouse anti rat phycoerythrin brdu monoclonal antibody igg
    H4K16Ac levels peak at S phase and drop dramatically in the G 2 /M transition. ( A ) Mammalian fibroblasts were pulsed with <t>BrdU,</t> stained with H4K16Ac antibodies as indicated, and analyzed by immunofluorescence for colocalization of BrdU and H4K16Ac. Two different samples are shown (Exp1 and Exp2). ( B ) Same cells as in A were pulsed with BrdU, incubated with H4K16Ac antibody and 7AAD (DNA content marker), and analyzed by FACS for the levels of H4K16Ac at each stage of the cell cycle. ( Upper ). Distribution of H4K16Ac levels in cells at each stage of the cell cycle is shown graphically and numerically, with 100% representing the levels of H4K16Ac in G 1 phase. ( C ) Immunofluorescence as in A, but in this case with the mitotic marker H3S28P and H4K16Ac.
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    Becton Dickinson fitc conjugated mouse monoclonal class igg1 anti brdu antibody
    H4K16Ac levels peak at S phase and drop dramatically in the G 2 /M transition. ( A ) Mammalian fibroblasts were pulsed with <t>BrdU,</t> stained with H4K16Ac antibodies as indicated, and analyzed by immunofluorescence for colocalization of BrdU and H4K16Ac. Two different samples are shown (Exp1 and Exp2). ( B ) Same cells as in A were pulsed with BrdU, incubated with H4K16Ac antibody and 7AAD (DNA content marker), and analyzed by FACS for the levels of H4K16Ac at each stage of the cell cycle. ( Upper ). Distribution of H4K16Ac levels in cells at each stage of the cell cycle is shown graphically and numerically, with 100% representing the levels of H4K16Ac in G 1 phase. ( C ) Immunofluorescence as in A, but in this case with the mitotic marker H3S28P and H4K16Ac.
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    Becton Dickinson fitc conjugated anti brdu monoclonal antibody
    H4K16Ac levels peak at S phase and drop dramatically in the G 2 /M transition. ( A ) Mammalian fibroblasts were pulsed with <t>BrdU,</t> stained with H4K16Ac antibodies as indicated, and analyzed by immunofluorescence for colocalization of BrdU and H4K16Ac. Two different samples are shown (Exp1 and Exp2). ( B ) Same cells as in A were pulsed with BrdU, incubated with H4K16Ac antibody and 7AAD (DNA content marker), and analyzed by FACS for the levels of H4K16Ac at each stage of the cell cycle. ( Upper ). Distribution of H4K16Ac levels in cells at each stage of the cell cycle is shown graphically and numerically, with 100% representing the levels of H4K16Ac in G 1 phase. ( C ) Immunofluorescence as in A, but in this case with the mitotic marker H3S28P and H4K16Ac.
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    Becton Dickinson fitc labeled anti brdu monoclonal antibody
    H4K16Ac levels peak at S phase and drop dramatically in the G 2 /M transition. ( A ) Mammalian fibroblasts were pulsed with <t>BrdU,</t> stained with H4K16Ac antibodies as indicated, and analyzed by immunofluorescence for colocalization of BrdU and H4K16Ac. Two different samples are shown (Exp1 and Exp2). ( B ) Same cells as in A were pulsed with BrdU, incubated with H4K16Ac antibody and 7AAD (DNA content marker), and analyzed by FACS for the levels of H4K16Ac at each stage of the cell cycle. ( Upper ). Distribution of H4K16Ac levels in cells at each stage of the cell cycle is shown graphically and numerically, with 100% representing the levels of H4K16Ac in G 1 phase. ( C ) Immunofluorescence as in A, but in this case with the mitotic marker H3S28P and H4K16Ac.
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    Becton Dickinson mouse monoclonal anti cdc42
    Increase in cell proliferation, apoptosis, and fibrosis in kidneys of <t>Cdc42</t> conditional knockout mice. (A and B) BrdU incorporation, a marker of active cell division, is significantly increased in sections of kidneys from the Cdc42 kidney-specific knockout
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    Becton Dickinson rat anti mouse cd41 monoclonal antibody
    Pulmonary accumulation of platelets carrying stromal cell-derived factor 1 (SDF1/CXCL12) after PNX a ) Immunostaining of SDF1, VE-cadherin, and platelet marker in mouse lung cryosections. <t>CD41</t> + SDF-1 + platelets were associated with VE-cadherin + PCECs after PNX (inset). Scale bar = 50 μm. b, c ) Flow cytometry analysis of SDF1 + platelet recruitment in the remaining lungs after PNX. Red color depicts SDF1 + CD41 + platelets, and small amounts of CD41 − SDF1 + cells are presented in yellow color. Kinetics of platelet accumulation in the lungs after PNX is assessed (c). n = 6 animals in all time points. d-f ) Mouse alveolar regrowth in Thpo −/− mice after injection of recombinant SDF1. Thpo −/− mice were treated with SDF1 injection, and AEC1 distribution (e) and gas exchange function (f) treated mice was assessed by comparing with vehicle-injected group. n =4 mice in both groups; P = 0.0055 between two groups; Scale bar = 50 μm. g-i ) Inducible deletion of Sdf1 in adult mice ( Sdf1 Δ/Δ ). Mice expressing tamoxifen-responsive Rosa-Cre ERT2 were crossed with Sdf1 loxP/loxP mice to generate Sdf1 Δ/Δ and control Sdf1 Δ/+ mice. SDF1 protein was determined by immunoblot in isolated platelets from mice 30 days after last injection. Protein level in indicated groups was compared after normalization to β-actin; Representative image is shown (i). SDF1 protein in Sdf1 Δ/Δ platelets is 3% of that of wild type mice (n = 6 mice in all genotypes). j-k ) Proliferation of AEC2s (j) and lung regrowth (k) in Sdf1 Δ/Δ and control mice after PNX. (k): n = 4 and 3 in sham-operated control and Sdf1 Δ/Δ mice, n = 5 and 4 in control and Sdf1 Δ/Δ mice group that underwent PNX. Scale bar = 50 μm. Error bar depicts s.e.m., and line stands for mean for panels c, f, h, k. Statistical difference between groups was assessed by unpaired two tail t-test.
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    Becton Dickinson anti mouse siglec f mab
    In mice lacking CCR2, the accumulation of Mφs and M2s in the colon postinfection is greatly reduced. CCR2 −/− and WT control mice (C57BL/6) were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of Mφs (F4/80 + cells) was conducted on sections of the proximal colon. Representative photographs of the F4/80 staining are shown in ( A ), and the quantitative analysis is shown in ( B ). Immunohistochemical staining of M2s (RELMα + cells) was also performed on sections of the proximal colon. Representative photographs of the RELMα staining are shown in ( C ), and the quantitative analysis is shown in ( D ). Scale bars, 100 μm. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Ly6G − <t>Siglec-F</t> − cells (as shown in E ). Representative plots of RELMα staining are shown in ( F ), and the data are shown graphically in ( G ). The values are the means ± SEM of five mice in each group. * p
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    Cell viability and proliferation on different SF/P(LLA-CL) membranes. Notes: The first row of ( A ) shows the results of the live/dead kit test, and few dead cells can be seen. The second row of ( A ) shows <t>Ki-67</t> protein staining, and the third row of ( A ) shows BrdU staining under laser scanning confocal microscopy. The scale bar indicates 50 μm in ( A ). The histogram of the positive percentage of Ki-67 protein staining is shown in ( B ). The histogram of the positive percentage of BrdU staining is shown in ( C ). * P
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    Becton Dickinson mouse monoclonal anti rat cd31
    PLC treatment reduces inflammation and endothelial dysfunction in rat TNBS-induced acute colitis. Representative microphotographs and morphometric evaluation of ICAM-1, VCAM-1, iNOS, <t>CD31,</t> PlGF, and BrDU staining of rat colon tissue from the different experimental groups: vehicle (50% ethanol, v/v), PLC (25 mg/kg intrarectal, twice daily for 1 week), TNBS and TNBS plus PLC (25 mg/kg intrarectal, twice daily for 1 week). Student's t -test: *, **, and *** P
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    Becton Dickinson mouse monoclonal anti β catenin
    Functional and structural analysis of the interactions between Sox9 and <t>β-catenin.</t> ( A ) Schematic representation of the β -catenin deletion mutants. ( B ) In vitro binding of β -catenin deletion mutants to 6xHis-tagged Sox9 bound to a nickel-resin. (I) Input; (B) bound to resin containing 6xHis-tagged Sox9. ( C ) Activation of TOPFLASH in C3H10T1/2 cells by Tcf3 is inhibited by Sox9 in a dose-dependent manner. ( D ) Sox9 inhibits binding of Tcf3 to β-catenin in vitro. Sox9 and 35 S-labeled Tcf3 proteins are incubated with 200 ng of 6xHis-tagged β-catenin immobilized to a nickel-resin. Of the input Tcf3 protein (lane 1 ) and bound Tcf3 proteins (lanes 2 - 4 ), 10% are resolved on SDS-PAGE. ( E ) Binding between Sox9 and β-catenin in Cos-7 cells. Cos-7 cells are cotransfected with 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9 for 24 h. Cell lysates are incubated with anti-β-catenin antibody and protein G-agarose beads under gentle agitation for 3 h at 4°C. The beads are washed three times with buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1% Triton X-100), resuspended in 10 μL of SDS-polyacrylamide gel electrophoresis sample loading buffer, and boiled for 2 min. Western immunoblotting is then performed. Anti-HA:HRP monoclonal antibody (Roche) and anti-myc:HRP antibody (Invitrogen) are diluted 1:1000 and 1:5000, respectively.
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    Becton Dickinson anti mouse cd103 mab
    In mice lacking CCR2, the accumulation of Mφs and M2s in the colon postinfection is greatly reduced. CCR2 −/− and WT control mice (C57BL/6) were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of Mφs (F4/80 + cells) was conducted on sections of the proximal colon. Representative photographs of the F4/80 staining are shown in ( A ), and the quantitative analysis is shown in ( B ). Immunohistochemical staining of M2s (RELMα + cells) was also performed on sections of the proximal colon. Representative photographs of the RELMα staining are shown in ( C ), and the quantitative analysis is shown in ( D ). Scale bars, 100 μm. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + <t>CD103</t> − Ly6G − Siglec-F − cells (as shown in E ). Representative plots of RELMα staining are shown in ( F ), and the data are shown graphically in ( G ). The values are the means ± SEM of five mice in each group. * p
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    Becton Dickinson monoclonal mouse anti hif 1α
    Effects of <t>HIF</t> inhibitors on HPF cell proliferation. HPFs were treated with the <t>HIF-1α</t> inhibitor KC7F2 or the HIF-2α inhibitor TC-S 7009 and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. ( A , B ) Cell proliferation as determined by BrdU assay. Cells were incubated with BrdU for 12 hrs. ( C , D ) Cell viability as determined by LDH assay. Data were expressed as a percentage of control (0 µM inhibitor) for each oxygen condition. The absorbance of control at normoxia for proliferation was 0.39 ± 0.06 (n = 3) and for hypoxia was 0.66 ± 0.16 (n = 3). The viability (%) was calculated as 100% -cytotoxicity %. Cytotoxicity % = [(LDH activity of sample -Spontaneous LDH activity)/(Maximum LDH activity -Spontaneous LDH activity)] × 100. The absorbance of control at normoxia for LDH activity was 0.17 (n = 2) and for hypoxia was 0.16 (n = 2). Maximum and spontaneous LDH activity values for normoxia were 0.26 and 0.17 (n = 2), respectively. Maximum and spontaneous LDH activity values for hypoxia were 0.27 and 0.17 (n = 2), respectively. Values represent means ± SE for proliferation and means for viability.
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    Image Search Results


    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

    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

    Localization of PARP-1 to the replicating genomic region. (A) Digoxigenin–deoxy-UTP was introduced into m5S cells to label replicating cells. PARP-1 and digoxigenin–deoxy-UTP (dig-dUTP) were visualized by immunofluorescence. DNA was counterstained with DAPI. (B and C) Localization of PARP-1–EYFP and PCNA-RFP or PARP-1–EYFP and Topo I–DsRed during S phase in COS-7 cells. After pulse labeling with BrdU, cells were fixed and BrdU was immunodetected. (D) Typical localization of PARP-1–EYFP out of S phase in COS-7 cells. (E) Localization of PARP-1–EYFP and PCNA-RFP during S phase in HeLa cells. (F) Typical localization of PARP-1–EYFP out of S phase in HeLa cells. Overlaid images and magnified views of the boxed areas are shown. n, nucleoli. Bars, 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

    doi: 10.1083/jcb.200806068

    Figure Lengend Snippet: Localization of PARP-1 to the replicating genomic region. (A) Digoxigenin–deoxy-UTP was introduced into m5S cells to label replicating cells. PARP-1 and digoxigenin–deoxy-UTP (dig-dUTP) were visualized by immunofluorescence. DNA was counterstained with DAPI. (B and C) Localization of PARP-1–EYFP and PCNA-RFP or PARP-1–EYFP and Topo I–DsRed during S phase in COS-7 cells. After pulse labeling with BrdU, cells were fixed and BrdU was immunodetected. (D) Typical localization of PARP-1–EYFP out of S phase in COS-7 cells. (E) Localization of PARP-1–EYFP and PCNA-RFP during S phase in HeLa cells. (F) Typical localization of PARP-1–EYFP out of S phase in HeLa cells. Overlaid images and magnified views of the boxed areas are shown. n, nucleoli. Bars, 5 μm.

    Article Snippet: Combed DNA molecules were heat denatured in 50% formamide (Roche) and 2× SSC at 72°C for 12 min. For immunodetection of IdU- and CldU-labeled DNA, denatured DNA molecules were incubated with mouse anti-BrdU monoclonal antibody (1:5; BD) and rat anti-BrdU monoclonal antibody (1:25; Oxford Biotechnology) for 1 h at 37°C.

    Techniques: Immunofluorescence, Labeling

    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

    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

    BrdU-positive mossy cell precursors are decreased in the hilus of developing Rac1 N /Rac3 KO mice. Mice were injected twice with BrdU at E12.5–E13 as detailed in the Materials and Methods . BrdU-injected mice were then sacrificed at P8 or P13. ( A ) Sagittal sections of the dorsal hippocampus from P8 Rac3 KO (left panels) and Rac1 N /Rac3 KO (right panels) mice were immunostained with antibodies for BrdU (red) and for Prox1 (green); nuclei were visualized by DAPI (blue). GCL, granule cell layer; hi, hilus. Scale bar: 50 µm. ( B ) Enlargement of the hilar region from the control section shown in ( A ). Arrows point to putative mossy cell precursors identified as BrdU-positive, Prox1-negative cells with a large round nucleus. Arrowheads indicate Prox1-positive granule cells. Scale bar: 12.5 µm. ( C ) Hippocampus from a P8 control mouse stained for BrdU. The areas used for quantification are indicated: zone 1 (Z1) includes the ventricular and subventricular zones and the dentate migratory stream; zone 2 (Z2) includes the hilar region, from which the zone that may include pyramidal cells from the CA3 has been excluded. Scale bar: 50 µm. ( D ) Quantification of BrdU-labelled cells in zone 1 and zone 2 of the hippocampus of Rac3 KO and Rac1 N /Rac3 KO littermates at P8 and P13. Bars are means ± SEM of the number of BrdU-positive cells (zone 1), and of BrdU-positive, Prox1-negative cells with large round nuclei (zone 2). At each stage, BrdU-positive cells were counted from 24–27 sections taken from 3 different mice per genotype. **P

    Journal: PLoS ONE

    Article Title: Rac1 and Rac3 GTPases Regulate the Development of Hilar Mossy Cells by Affecting the Migration of Their Precursors to the Hilus

    doi: 10.1371/journal.pone.0024819

    Figure Lengend Snippet: BrdU-positive mossy cell precursors are decreased in the hilus of developing Rac1 N /Rac3 KO mice. Mice were injected twice with BrdU at E12.5–E13 as detailed in the Materials and Methods . BrdU-injected mice were then sacrificed at P8 or P13. ( A ) Sagittal sections of the dorsal hippocampus from P8 Rac3 KO (left panels) and Rac1 N /Rac3 KO (right panels) mice were immunostained with antibodies for BrdU (red) and for Prox1 (green); nuclei were visualized by DAPI (blue). GCL, granule cell layer; hi, hilus. Scale bar: 50 µm. ( B ) Enlargement of the hilar region from the control section shown in ( A ). Arrows point to putative mossy cell precursors identified as BrdU-positive, Prox1-negative cells with a large round nucleus. Arrowheads indicate Prox1-positive granule cells. Scale bar: 12.5 µm. ( C ) Hippocampus from a P8 control mouse stained for BrdU. The areas used for quantification are indicated: zone 1 (Z1) includes the ventricular and subventricular zones and the dentate migratory stream; zone 2 (Z2) includes the hilar region, from which the zone that may include pyramidal cells from the CA3 has been excluded. Scale bar: 50 µm. ( D ) Quantification of BrdU-labelled cells in zone 1 and zone 2 of the hippocampus of Rac3 KO and Rac1 N /Rac3 KO littermates at P8 and P13. Bars are means ± SEM of the number of BrdU-positive cells (zone 1), and of BrdU-positive, Prox1-negative cells with large round nuclei (zone 2). At each stage, BrdU-positive cells were counted from 24–27 sections taken from 3 different mice per genotype. **P

    Article Snippet: Antibodies and plasmids The following antibodies and dilutions were used in this study: rabbit pAb anti-GluR2/3, 1∶100 for immunohistochemistry, 1∶50 for immunofluorescence (Upstate); mouse mAb anti-MAP2 (microtubule associated protein 2), 1∶250 (clone HM-2, Sigma-Aldrich); mouse mAb anti-Synaptotagmin 1, 1∶200 (Synaptic Systems); rabbit pAb anti-Prox1 (prospero-related homeobox 1), 1∶1000 (Millipore); mouse mAb anti-BrdU, 1∶100 (BD); rabbit pAb anti-zinc transporter-3 (ZnT-3), 1∶200 (gift of Richard Palmiter) .

    Techniques: Mouse Assay, Injection, Staining

    Spry1-2 −/− Shows Increased Proliferation, Precocious Progenitor Cell Maturation and Neurogenesis at E12.5 (A-C') Immunofluorescence analysis for Blbp (A-B', red), and Nestin (A-A' and C-C', green) in the WT and Spry1-2 −/− brains. Blbp, a marker for radial glia, is strongly up-regulated in the dorsal/medial pallium (arrowheads in B'). (D-D') Short-pulse BrdU labeling of WT (D) and Spry1-2 −/− (D'). BrdU was injected to pregnant females and E12.5 embryos were collected after 30', and coronal sections were stained with anti-BrdU antibody. More BrdU + cells are present in the dorsal pallium in Spry1-2 −/− cortex (D') than in WT (D). (E-E') RNA in situ hybridization for Tbr2 in WT and Spry1-2 −/− brains, showing the increase of Tbr2 staining in preplate (red arrowhead) and in VZ/SVZ region (black arrowhead). (F-F') RNA in situ hybridization for Etv1 in WT and Spry1-2 −/− brains, showing the increase of Etv1 staining in the preplate (arrowheads). (G-H') β-tubulin (green) and Blbp (red) immunodetection in WT and Spry1-2 −/− brains. Arrowheads in H' point to increase preplate thickness in Spry1-2 −/− null mice, in a region of high Blbp expression (H'). Scale bars: (A-C'), (F-G'), (H-I') 200 μm, (D-D'), 250 μm.

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

    Article Title: Repression of Fgf-Signaling by Sprouty1-2 Regulates Cortical Patterning in Two Distinct Regions and Times

    doi: 10.1523/JNEUROSCI.0307-10.2010

    Figure Lengend Snippet: Spry1-2 −/− Shows Increased Proliferation, Precocious Progenitor Cell Maturation and Neurogenesis at E12.5 (A-C') Immunofluorescence analysis for Blbp (A-B', red), and Nestin (A-A' and C-C', green) in the WT and Spry1-2 −/− brains. Blbp, a marker for radial glia, is strongly up-regulated in the dorsal/medial pallium (arrowheads in B'). (D-D') Short-pulse BrdU labeling of WT (D) and Spry1-2 −/− (D'). BrdU was injected to pregnant females and E12.5 embryos were collected after 30', and coronal sections were stained with anti-BrdU antibody. More BrdU + cells are present in the dorsal pallium in Spry1-2 −/− cortex (D') than in WT (D). (E-E') RNA in situ hybridization for Tbr2 in WT and Spry1-2 −/− brains, showing the increase of Tbr2 staining in preplate (red arrowhead) and in VZ/SVZ region (black arrowhead). (F-F') RNA in situ hybridization for Etv1 in WT and Spry1-2 −/− brains, showing the increase of Etv1 staining in the preplate (arrowheads). (G-H') β-tubulin (green) and Blbp (red) immunodetection in WT and Spry1-2 −/− brains. Arrowheads in H' point to increase preplate thickness in Spry1-2 −/− null mice, in a region of high Blbp expression (H'). Scale bars: (A-C'), (F-G'), (H-I') 200 μm, (D-D'), 250 μm.

    Article Snippet: The antibodies used were as follows: monoclonal anti-βIII-tubulin antibody (clone TUJ1; Covance), 1:1000; monoclonal anti-BrdU antibody (clone B44; Becton Dickinson), 1:500; anti-phospho-p44/42 Map Kinase (Thr202/Tyr204) antibody (Cell Signaling) 1:100; anti-phospho-Histone H3 (Ser10) (Upstate) 1:200; mouse anti-human Coup-TFI (clone H8132, Invitrogen) 1:1000; rabbit anti-Blbp (Chemicon) 1:1000 For fluorescent immunohistochemistry, goat anti-rabbit Alexa-488, goat anti-mouse Alexa-594 or goat anti-rat Alexa-594 antibodies (Molecular Probes, Eugene, OR), diluted at 1:300, were used.

    Techniques: Immunofluorescence, Marker, Labeling, Injection, Staining, RNA In Situ Hybridization, Immunodetection, Mouse Assay, Expressing

    Deletion of Irs2 suppresses epithelial growth, proliferation, and Myc. A: Proliferation index (PI) determined by BrdU immunolabeling of PIN. PI is significantly reduced in Pten +/− Irs2 −/− mice ( P

    Journal: The American Journal of Pathology

    Article Title: Irs2 Inactivation Suppresses Tumor Progression in Pten+/− Mice

    doi: 10.2353/ajpath.2009.080086

    Figure Lengend Snippet: Deletion of Irs2 suppresses epithelial growth, proliferation, and Myc. A: Proliferation index (PI) determined by BrdU immunolabeling of PIN. PI is significantly reduced in Pten +/− Irs2 −/− mice ( P

    Article Snippet: Antibodies (Abs) used include anti-IRS2 polyclonal Ab (1:50; Upstate, Charlottesville, VA), anti-Pten monoclonal Ab (1:200; Cell Signaling Technology, Danvers, MA), anti-BrdU monoclonal Ab (1:100; BD Biosciences, San Jose, CA), anti-phosphoAkt (Ser473) monoclonal Ab (1:100, Cell Signaling Technology), anti-phosphoS6 (Ser235/236) ribosomal protein polyclonal Ab (1:50, Cell Signaling Technology), anti-phospho Erk1,2 (Thr202/Tyr204) monoclonal Ab (1:100, Cell Signaling Technology), anti-Myc polyclonal Ab (N-262) (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-E-cadherin monoclonal Ab (1:100; Calbiochem, San Diego, CA), anti-cytokeratin monoclonal Abs (AE1/AE3) (1:50; Chemicon Int.

    Techniques: Immunolabeling, Mouse Assay

    Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and CldU (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with BrdU at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p

    Journal: The Journal of Neuroscience

    Article Title: Stimulation of Entorhinal Cortex Promotes Adult Neurogenesis and Facilitates Spatial Memory

    doi: 10.1523/JNEUROSCI.3100-11.2011

    Figure Lengend Snippet: Survival and differentiation of stimulation-induced neurons. a , After unilateral stimulation ( n = 5), mice were injected with IdU (during the period of increased proliferation) and CldU (once proliferation returned to baseline). b , Representative confocal image of IdU + and CldU + DG cells colabeled with NeuN (scale bar, 20 μm), ipsilateral to stimulation. c , Similar proportions of IdU + and CldU + cells were NeuN + ipsilateral (I) and contralateral (C) to electrode site. d , Separate groups of mice were injected with BrdU at different delays before stimulation ( n = 8 per group). e , There were more BrdU + cells ipsilateral to the stimulation site in the mice treated with BrdU 10 d before surgery. ** p

    Article Snippet: For other analyses, the following primary antibodies were used: rabbit polyclonal anti-Fos (1:1000; Calbiochem), rat monoclonal anti-BrdU for BrdU and CldU specifically (1:500; Accurate Chemicals), mouse monoclonal anti-BrdU for IdU specifically (1:1000; BD Biosciences), mouse monoclonal anti-NeuN (1:1000; Millipore Bioscience Research Reagents), rabbit polyclonal anti-GFP (1:500; Invitrogen), and Alexa Fluor 488-conjugated mouse monoclonal anti-NeuN (1:1000; Millipore Bioscience Research Reagents).

    Techniques: Mouse Assay, Injection

    Proliferation of CD4 + FoxP3 + Treg population after ablative conditioning . B6 mice were subjected to 9.5 Gy TBI. On the following day, mice received 5 × 10 6 T-depleted BM cells isolated from Thy1.1 congenic B6 mice. Mice were killed 20, 35, and 56 days post-BMT and spleen and lymph nodes subjected to FAC analysis. Four days before death, mice were placed on BrdU (0.8 mg/mL) drinking water. (A) Representative dot plot showing the Thy1.1 (donor BM) staining and IgG of BrdU staining of the gated CD4 + Foxp3 + cells in the spleen (left panels) and lymph nodes (right panels). (B) Percentage BrdU + cells in the host (Thy1.1 − ) and donor (Thy1.1 + ) CD4 + Foxp3 + and CD4 + Foxp3 − compartments in the spleen (left) and lymph nodes (right). (C) Wild-type B6 mice (n = 3) were placed on BrdU drinking water for 4 days, killed, and the spleen and lymph nodes subjected to FACS analysis. Representative dot plot showing CD4 and Foxp3 expression with corresponding histograms illustrating staining by isotype control (IgG) and anti-BrdU mAb of the gated CD4 + FoxP3 + and CD4 + Foxp3 − populations in the spleen (left) and lymph nodes (right). Data in panel B are mean plus or minus SEM for 3 mice per group per time point.

    Journal: Blood

    Article Title: Host CD4+CD25+ T cells can expand and comprise a major component of the Treg compartment after experimental HCT

    doi: 10.1182/blood-2008-08-173179

    Figure Lengend Snippet: Proliferation of CD4 + FoxP3 + Treg population after ablative conditioning . B6 mice were subjected to 9.5 Gy TBI. On the following day, mice received 5 × 10 6 T-depleted BM cells isolated from Thy1.1 congenic B6 mice. Mice were killed 20, 35, and 56 days post-BMT and spleen and lymph nodes subjected to FAC analysis. Four days before death, mice were placed on BrdU (0.8 mg/mL) drinking water. (A) Representative dot plot showing the Thy1.1 (donor BM) staining and IgG of BrdU staining of the gated CD4 + Foxp3 + cells in the spleen (left panels) and lymph nodes (right panels). (B) Percentage BrdU + cells in the host (Thy1.1 − ) and donor (Thy1.1 + ) CD4 + Foxp3 + and CD4 + Foxp3 − compartments in the spleen (left) and lymph nodes (right). (C) Wild-type B6 mice (n = 3) were placed on BrdU drinking water for 4 days, killed, and the spleen and lymph nodes subjected to FACS analysis. Representative dot plot showing CD4 and Foxp3 expression with corresponding histograms illustrating staining by isotype control (IgG) and anti-BrdU mAb of the gated CD4 + FoxP3 + and CD4 + Foxp3 − populations in the spleen (left) and lymph nodes (right). Data in panel B are mean plus or minus SEM for 3 mice per group per time point.

    Article Snippet: The following antibodies were used for flow cytometric analysis: anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD25 (PC61), anti-Thy1.1 (OX-7), anti-CD45.1 (A.20), anti-Thy1.2 (53-2.1), anti-BrdU monoclonal antibodies (mAbs) obtained from BD Biosciences (San Jose, CA) and anti-Foxp3 (FJK16s) mAb from eBioscience (San Diego, CA).

    Techniques: Mouse Assay, Isolation, Staining, BrdU Staining, FACS, Expressing

    H4K16Ac levels peak at S phase and drop dramatically in the G 2 /M transition. ( A ) Mammalian fibroblasts were pulsed with BrdU, stained with H4K16Ac antibodies as indicated, and analyzed by immunofluorescence for colocalization of BrdU and H4K16Ac. Two different samples are shown (Exp1 and Exp2). ( B ) Same cells as in A were pulsed with BrdU, incubated with H4K16Ac antibody and 7AAD (DNA content marker), and analyzed by FACS for the levels of H4K16Ac at each stage of the cell cycle. ( Upper ). Distribution of H4K16Ac levels in cells at each stage of the cell cycle is shown graphically and numerically, with 100% representing the levels of H4K16Ac in G 1 phase. ( C ) Immunofluorescence as in A, but in this case with the mitotic marker H3S28P and H4K16Ac.

    Journal: Genes & Development

    Article Title: SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis

    doi: 10.1101/gad.1412706

    Figure Lengend Snippet: H4K16Ac levels peak at S phase and drop dramatically in the G 2 /M transition. ( A ) Mammalian fibroblasts were pulsed with BrdU, stained with H4K16Ac antibodies as indicated, and analyzed by immunofluorescence for colocalization of BrdU and H4K16Ac. Two different samples are shown (Exp1 and Exp2). ( B ) Same cells as in A were pulsed with BrdU, incubated with H4K16Ac antibody and 7AAD (DNA content marker), and analyzed by FACS for the levels of H4K16Ac at each stage of the cell cycle. ( Upper ). Distribution of H4K16Ac levels in cells at each stage of the cell cycle is shown graphically and numerically, with 100% representing the levels of H4K16Ac in G 1 phase. ( C ) Immunofluorescence as in A, but in this case with the mitotic marker H3S28P and H4K16Ac.

    Article Snippet: After washing, anti-rabbit PE secondary antibody was added (Molecular Probes) in combination with anti-BrdU fluorescein-conjugated mouse monoclonal antibody (BD Pharmigen) for 30 min. DNA was counterstained using 7AAD fluorescent dye (BD Biosciences).

    Techniques: Staining, Immunofluorescence, Incubation, Marker, FACS

    Increase in cell proliferation, apoptosis, and fibrosis in kidneys of Cdc42 conditional knockout mice. (A and B) BrdU incorporation, a marker of active cell division, is significantly increased in sections of kidneys from the Cdc42 kidney-specific knockout

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

    doi: 10.1681/ASN.2012121236

    Figure Lengend Snippet: Increase in cell proliferation, apoptosis, and fibrosis in kidneys of Cdc42 conditional knockout mice. (A and B) BrdU incorporation, a marker of active cell division, is significantly increased in sections of kidneys from the Cdc42 kidney-specific knockout

    Article Snippet: The antibodies used in this study were mouse monoclonal anti-Cdc42 (610929, BD Transduction Laboratories), rabbit polyclonal anti–phospho-ERK1/2 (#9101, Cell Signaling), rabbit polyclonal anti-total ERK1(/2) (#9102, Cell Signaling), mouse monoclonal anti–glyceraldehyde 3-phosphate dehydrogenase (G8795, Sigma), rabbit polyclonal anti-γ tubulin (T5192, Sigma), mouse monoclonal antiacetylated α-tubulin (T6793, Sigma), rat monoclonal anti-BrdU (ab6326, Abcam), mouse anti-GP135 (a gift from Dr. George Ojakian, State University of New York), rabbit anti–E-cadherin (ab15148, Abcam), biotinylated LTA (B-1325, Vector Laboratories), biotinylated PNA (B-1075, Vector Laboratories).

    Techniques: Knock-Out, Mouse Assay, BrdU Incorporation Assay, Marker

    Active pERK is increased in the kidneys of Cdc42 kidney-specific knockout mice. (A) Immunoblot analysis of proteins extracted from the whole kidneys of P6 control and Cdc42 kidney-specific knockout mice, showed increased pERK levels in Cdc42 knockout

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

    doi: 10.1681/ASN.2012121236

    Figure Lengend Snippet: Active pERK is increased in the kidneys of Cdc42 kidney-specific knockout mice. (A) Immunoblot analysis of proteins extracted from the whole kidneys of P6 control and Cdc42 kidney-specific knockout mice, showed increased pERK levels in Cdc42 knockout

    Article Snippet: The antibodies used in this study were mouse monoclonal anti-Cdc42 (610929, BD Transduction Laboratories), rabbit polyclonal anti–phospho-ERK1/2 (#9101, Cell Signaling), rabbit polyclonal anti-total ERK1(/2) (#9102, Cell Signaling), mouse monoclonal anti–glyceraldehyde 3-phosphate dehydrogenase (G8795, Sigma), rabbit polyclonal anti-γ tubulin (T5192, Sigma), mouse monoclonal antiacetylated α-tubulin (T6793, Sigma), rat monoclonal anti-BrdU (ab6326, Abcam), mouse anti-GP135 (a gift from Dr. George Ojakian, State University of New York), rabbit anti–E-cadherin (ab15148, Abcam), biotinylated LTA (B-1325, Vector Laboratories), biotinylated PNA (B-1075, Vector Laboratories).

    Techniques: Knock-Out, Mouse Assay

    Primary ciliogenesis is inhibited in the cysts of Cdc42 kidney-specific knockout mice. (A and B) Immunostaining of acetylated α-tubulin (red) in kidneys from control (A) and Cdc42 kidney-specific knockout mice (B) at P4, shows lack of cilia in

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

    doi: 10.1681/ASN.2012121236

    Figure Lengend Snippet: Primary ciliogenesis is inhibited in the cysts of Cdc42 kidney-specific knockout mice. (A and B) Immunostaining of acetylated α-tubulin (red) in kidneys from control (A) and Cdc42 kidney-specific knockout mice (B) at P4, shows lack of cilia in

    Article Snippet: The antibodies used in this study were mouse monoclonal anti-Cdc42 (610929, BD Transduction Laboratories), rabbit polyclonal anti–phospho-ERK1/2 (#9101, Cell Signaling), rabbit polyclonal anti-total ERK1(/2) (#9102, Cell Signaling), mouse monoclonal anti–glyceraldehyde 3-phosphate dehydrogenase (G8795, Sigma), rabbit polyclonal anti-γ tubulin (T5192, Sigma), mouse monoclonal antiacetylated α-tubulin (T6793, Sigma), rat monoclonal anti-BrdU (ab6326, Abcam), mouse anti-GP135 (a gift from Dr. George Ojakian, State University of New York), rabbit anti–E-cadherin (ab15148, Abcam), biotinylated LTA (B-1325, Vector Laboratories), biotinylated PNA (B-1075, Vector Laboratories).

    Techniques: Knock-Out, Mouse Assay, Immunostaining

    Lack of Cdc42 in kidney tubule cells leads to an early postnatal death. KspCre;Cdc42 fl/+ mice were mated to Cdc42 fl/fl mice as illustrated in . Of 65 pups tested from E16.5 to P10, there were 15 Cdc42 fl/fl , 25 Cdc42 fl/+ , 12 KspCre;Cdc42 fl/+ ,

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

    doi: 10.1681/ASN.2012121236

    Figure Lengend Snippet: Lack of Cdc42 in kidney tubule cells leads to an early postnatal death. KspCre;Cdc42 fl/+ mice were mated to Cdc42 fl/fl mice as illustrated in . Of 65 pups tested from E16.5 to P10, there were 15 Cdc42 fl/fl , 25 Cdc42 fl/+ , 12 KspCre;Cdc42 fl/+ ,

    Article Snippet: The antibodies used in this study were mouse monoclonal anti-Cdc42 (610929, BD Transduction Laboratories), rabbit polyclonal anti–phospho-ERK1/2 (#9101, Cell Signaling), rabbit polyclonal anti-total ERK1(/2) (#9102, Cell Signaling), mouse monoclonal anti–glyceraldehyde 3-phosphate dehydrogenase (G8795, Sigma), rabbit polyclonal anti-γ tubulin (T5192, Sigma), mouse monoclonal antiacetylated α-tubulin (T6793, Sigma), rat monoclonal anti-BrdU (ab6326, Abcam), mouse anti-GP135 (a gift from Dr. George Ojakian, State University of New York), rabbit anti–E-cadherin (ab15148, Abcam), biotinylated LTA (B-1325, Vector Laboratories), biotinylated PNA (B-1075, Vector Laboratories).

    Techniques: Mouse Assay

    cdc42 and sec10 genetically interact. A synergistic interaction resulting in hydrocephalus (arrowhead), small eyes (arrow), pericardial edema (*), and tail defects was observed upon co-injection of suboptimal doses of 2 ng cdc42MO plus 7.5 ng sec10MO

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

    doi: 10.1681/ASN.2012121236

    Figure Lengend Snippet: cdc42 and sec10 genetically interact. A synergistic interaction resulting in hydrocephalus (arrowhead), small eyes (arrow), pericardial edema (*), and tail defects was observed upon co-injection of suboptimal doses of 2 ng cdc42MO plus 7.5 ng sec10MO

    Article Snippet: The antibodies used in this study were mouse monoclonal anti-Cdc42 (610929, BD Transduction Laboratories), rabbit polyclonal anti–phospho-ERK1/2 (#9101, Cell Signaling), rabbit polyclonal anti-total ERK1(/2) (#9102, Cell Signaling), mouse monoclonal anti–glyceraldehyde 3-phosphate dehydrogenase (G8795, Sigma), rabbit polyclonal anti-γ tubulin (T5192, Sigma), mouse monoclonal antiacetylated α-tubulin (T6793, Sigma), rat monoclonal anti-BrdU (ab6326, Abcam), mouse anti-GP135 (a gift from Dr. George Ojakian, State University of New York), rabbit anti–E-cadherin (ab15148, Abcam), biotinylated LTA (B-1325, Vector Laboratories), biotinylated PNA (B-1075, Vector Laboratories).

    Techniques: Injection

    Kidney tubule cell-specific Cdc42 knockout mice were successfully generated. (A) Targeting scheme showing the Cdc42 gene (wild type), the targeting construct, and the conditional allele after homologous recombination of the targeting construct and removal

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

    doi: 10.1681/ASN.2012121236

    Figure Lengend Snippet: Kidney tubule cell-specific Cdc42 knockout mice were successfully generated. (A) Targeting scheme showing the Cdc42 gene (wild type), the targeting construct, and the conditional allele after homologous recombination of the targeting construct and removal

    Article Snippet: The antibodies used in this study were mouse monoclonal anti-Cdc42 (610929, BD Transduction Laboratories), rabbit polyclonal anti–phospho-ERK1/2 (#9101, Cell Signaling), rabbit polyclonal anti-total ERK1(/2) (#9102, Cell Signaling), mouse monoclonal anti–glyceraldehyde 3-phosphate dehydrogenase (G8795, Sigma), rabbit polyclonal anti-γ tubulin (T5192, Sigma), mouse monoclonal antiacetylated α-tubulin (T6793, Sigma), rat monoclonal anti-BrdU (ab6326, Abcam), mouse anti-GP135 (a gift from Dr. George Ojakian, State University of New York), rabbit anti–E-cadherin (ab15148, Abcam), biotinylated LTA (B-1325, Vector Laboratories), biotinylated PNA (B-1075, Vector Laboratories).

    Techniques: Knock-Out, Mouse Assay, Generated, Construct, Homologous Recombination

    cdc42 expression occurs in the zebrafish kidney, eye, and brain, and cdc42 knockdown by antisense MOs results in abnormal phenotypes. (A) Lateral views of whole mount in situ hybridization of zebrafish embryos at 3 dpf with antisense (upper part) and

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

    doi: 10.1681/ASN.2012121236

    Figure Lengend Snippet: cdc42 expression occurs in the zebrafish kidney, eye, and brain, and cdc42 knockdown by antisense MOs results in abnormal phenotypes. (A) Lateral views of whole mount in situ hybridization of zebrafish embryos at 3 dpf with antisense (upper part) and

    Article Snippet: The antibodies used in this study were mouse monoclonal anti-Cdc42 (610929, BD Transduction Laboratories), rabbit polyclonal anti–phospho-ERK1/2 (#9101, Cell Signaling), rabbit polyclonal anti-total ERK1(/2) (#9102, Cell Signaling), mouse monoclonal anti–glyceraldehyde 3-phosphate dehydrogenase (G8795, Sigma), rabbit polyclonal anti-γ tubulin (T5192, Sigma), mouse monoclonal antiacetylated α-tubulin (T6793, Sigma), rat monoclonal anti-BrdU (ab6326, Abcam), mouse anti-GP135 (a gift from Dr. George Ojakian, State University of New York), rabbit anti–E-cadherin (ab15148, Abcam), biotinylated LTA (B-1325, Vector Laboratories), biotinylated PNA (B-1075, Vector Laboratories).

    Techniques: Expressing, In Situ Hybridization

    Model for the role of Cdc42 in delivery of ciliary proteins. (A) Our data support a model in which the exocyst complex is localized to the primary cilium by Cdc42, which is located all along the apical surface but is activated at the primary cilium by

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

    doi: 10.1681/ASN.2012121236

    Figure Lengend Snippet: Model for the role of Cdc42 in delivery of ciliary proteins. (A) Our data support a model in which the exocyst complex is localized to the primary cilium by Cdc42, which is located all along the apical surface but is activated at the primary cilium by

    Article Snippet: The antibodies used in this study were mouse monoclonal anti-Cdc42 (610929, BD Transduction Laboratories), rabbit polyclonal anti–phospho-ERK1/2 (#9101, Cell Signaling), rabbit polyclonal anti-total ERK1(/2) (#9102, Cell Signaling), mouse monoclonal anti–glyceraldehyde 3-phosphate dehydrogenase (G8795, Sigma), rabbit polyclonal anti-γ tubulin (T5192, Sigma), mouse monoclonal antiacetylated α-tubulin (T6793, Sigma), rat monoclonal anti-BrdU (ab6326, Abcam), mouse anti-GP135 (a gift from Dr. George Ojakian, State University of New York), rabbit anti–E-cadherin (ab15148, Abcam), biotinylated LTA (B-1325, Vector Laboratories), biotinylated PNA (B-1075, Vector Laboratories).

    Techniques:

    KspCre;Cdc42 fl/fl mice develop renal cysts in the distal and collecting tubules. (A–D) Hematoxylin and eosin–stained sections of kidneys from control (A and C) and Cdc42 kidney-specific knockout mice (B and D) at P4 (A and B) and P6 (C

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

    doi: 10.1681/ASN.2012121236

    Figure Lengend Snippet: KspCre;Cdc42 fl/fl mice develop renal cysts in the distal and collecting tubules. (A–D) Hematoxylin and eosin–stained sections of kidneys from control (A and C) and Cdc42 kidney-specific knockout mice (B and D) at P4 (A and B) and P6 (C

    Article Snippet: The antibodies used in this study were mouse monoclonal anti-Cdc42 (610929, BD Transduction Laboratories), rabbit polyclonal anti–phospho-ERK1/2 (#9101, Cell Signaling), rabbit polyclonal anti-total ERK1(/2) (#9102, Cell Signaling), mouse monoclonal anti–glyceraldehyde 3-phosphate dehydrogenase (G8795, Sigma), rabbit polyclonal anti-γ tubulin (T5192, Sigma), mouse monoclonal antiacetylated α-tubulin (T6793, Sigma), rat monoclonal anti-BrdU (ab6326, Abcam), mouse anti-GP135 (a gift from Dr. George Ojakian, State University of New York), rabbit anti–E-cadherin (ab15148, Abcam), biotinylated LTA (B-1325, Vector Laboratories), biotinylated PNA (B-1075, Vector Laboratories).

    Techniques: Mouse Assay, Staining, Knock-Out

    Pulmonary accumulation of platelets carrying stromal cell-derived factor 1 (SDF1/CXCL12) after PNX a ) Immunostaining of SDF1, VE-cadherin, and platelet marker in mouse lung cryosections. CD41 + SDF-1 + platelets were associated with VE-cadherin + PCECs after PNX (inset). Scale bar = 50 μm. b, c ) Flow cytometry analysis of SDF1 + platelet recruitment in the remaining lungs after PNX. Red color depicts SDF1 + CD41 + platelets, and small amounts of CD41 − SDF1 + cells are presented in yellow color. Kinetics of platelet accumulation in the lungs after PNX is assessed (c). n = 6 animals in all time points. d-f ) Mouse alveolar regrowth in Thpo −/− mice after injection of recombinant SDF1. Thpo −/− mice were treated with SDF1 injection, and AEC1 distribution (e) and gas exchange function (f) treated mice was assessed by comparing with vehicle-injected group. n =4 mice in both groups; P = 0.0055 between two groups; Scale bar = 50 μm. g-i ) Inducible deletion of Sdf1 in adult mice ( Sdf1 Δ/Δ ). Mice expressing tamoxifen-responsive Rosa-Cre ERT2 were crossed with Sdf1 loxP/loxP mice to generate Sdf1 Δ/Δ and control Sdf1 Δ/+ mice. SDF1 protein was determined by immunoblot in isolated platelets from mice 30 days after last injection. Protein level in indicated groups was compared after normalization to β-actin; Representative image is shown (i). SDF1 protein in Sdf1 Δ/Δ platelets is 3% of that of wild type mice (n = 6 mice in all genotypes). j-k ) Proliferation of AEC2s (j) and lung regrowth (k) in Sdf1 Δ/Δ and control mice after PNX. (k): n = 4 and 3 in sham-operated control and Sdf1 Δ/Δ mice, n = 5 and 4 in control and Sdf1 Δ/Δ mice group that underwent PNX. Scale bar = 50 μm. Error bar depicts s.e.m., and line stands for mean for panels c, f, h, k. Statistical difference between groups was assessed by unpaired two tail t-test.

    Journal: Nature cell biology

    Article Title: Platelet-derived SDF1 primes pulmonary capillary vascular niche to drive lung alveolar regeneration

    doi: 10.1038/ncb3096

    Figure Lengend Snippet: Pulmonary accumulation of platelets carrying stromal cell-derived factor 1 (SDF1/CXCL12) after PNX a ) Immunostaining of SDF1, VE-cadherin, and platelet marker in mouse lung cryosections. CD41 + SDF-1 + platelets were associated with VE-cadherin + PCECs after PNX (inset). Scale bar = 50 μm. b, c ) Flow cytometry analysis of SDF1 + platelet recruitment in the remaining lungs after PNX. Red color depicts SDF1 + CD41 + platelets, and small amounts of CD41 − SDF1 + cells are presented in yellow color. Kinetics of platelet accumulation in the lungs after PNX is assessed (c). n = 6 animals in all time points. d-f ) Mouse alveolar regrowth in Thpo −/− mice after injection of recombinant SDF1. Thpo −/− mice were treated with SDF1 injection, and AEC1 distribution (e) and gas exchange function (f) treated mice was assessed by comparing with vehicle-injected group. n =4 mice in both groups; P = 0.0055 between two groups; Scale bar = 50 μm. g-i ) Inducible deletion of Sdf1 in adult mice ( Sdf1 Δ/Δ ). Mice expressing tamoxifen-responsive Rosa-Cre ERT2 were crossed with Sdf1 loxP/loxP mice to generate Sdf1 Δ/Δ and control Sdf1 Δ/+ mice. SDF1 protein was determined by immunoblot in isolated platelets from mice 30 days after last injection. Protein level in indicated groups was compared after normalization to β-actin; Representative image is shown (i). SDF1 protein in Sdf1 Δ/Δ platelets is 3% of that of wild type mice (n = 6 mice in all genotypes). j-k ) Proliferation of AEC2s (j) and lung regrowth (k) in Sdf1 Δ/Δ and control mice after PNX. (k): n = 4 and 3 in sham-operated control and Sdf1 Δ/Δ mice, n = 5 and 4 in control and Sdf1 Δ/Δ mice group that underwent PNX. Scale bar = 50 μm. Error bar depicts s.e.m., and line stands for mean for panels c, f, h, k. Statistical difference between groups was assessed by unpaired two tail t-test.

    Article Snippet: To deplete circulating platelets, rat anti-mouse CD41 monoclonal antibody (BD Biosciences, clone MWReg30) was intraperitoneally injected into mice at dose of 10 mg/kg every three days.

    Techniques: Derivative Assay, Immunostaining, Marker, Flow Cytometry, Cytometry, Mouse Assay, Injection, Recombinant, Expressing, Isolation

    Intravascular transfusion of Sdf1 +/+ but not Sdf1 -deficient ( Sdf1 −/− ) platelets promotes alveolar regeneration in Thpo −/− mice a-c ) Platelets accumulate in the lungs of pneumonectomized Thpo −/− mice after intravascular infusion. Platelets were isolated from β-actin promoter driven- tdTomato ( ACTB-tdTomato ) mice and infused into Thpo −/− mice (a). Accumulation of tdTomato + CD41 + platelets in the lungs of Thpo −/− mice was determined by flow cytometry and immunostaining; Scale bar = 50 μm. d-g) Strategy to examine the influence of platelet-derived SDF1 on lung alveolar regeneration. Sdf1 +/+ and Sdf1 −/− platelets were isolated from wild type and Sdf1 Δ/Δ mice and infused into pneumonectomized Thpo −/− mice, respectively. Recovery of right lung volume (e), mass (f) and respiratory function (g) were determined in recipient mice. In panels (e) and (f), n =5 mice in all groups; P = 0.011 (e) and 0.026 (f) between mice transplanted with Sdf1 +/+ and Sdf1 −/− platelets. In panel (g), n = 6 mice ( Sdf1 +/+ ) and 4 mice ( Sdf1 −/− ) . P = 0.0013 (left) and 0.0042 (right) between two groups. h-k ) Expansion of AEC2s (h, j) and PCECs (i, k) in Thpo −/− mice after infusion of Sdf1 +/+ and Sdf1 −/− platelets. Representative immunostaining image of two transplanted groups and flow cytometry graph are shown; n = 5 mixw in all groups; P = 0.00011 (j) and 0.00081 (k) between two platelet types. Scale bar = 50 μm. l ) AEC1s in Thpo −/− mice following PNX and Sdf1 +/+ and Sdf1 −/− platelet transplantation. Scale bar = 50 μm. m-o ) Alveolar architecture of Thpo −/− mice receiving platelet infusion after PNX. Alveolar morphology was assessed by H E staining (m), and alveolar mean linear intercept (n) and number (o) were compared; n = 5 mice in both groups; P = 0.027 (n) and 0.0015 (o) between mice transplanted with Sdf1 +/+ and Sdf1 −/− platelets. Scale bar = 50 um. Error bar defines s.e.m., and line represents mean for panels e, f, g, j, k, n, o. Statistical difference between groups was assessed by unpaired two tail t-test.

    Journal: Nature cell biology

    Article Title: Platelet-derived SDF1 primes pulmonary capillary vascular niche to drive lung alveolar regeneration

    doi: 10.1038/ncb3096

    Figure Lengend Snippet: Intravascular transfusion of Sdf1 +/+ but not Sdf1 -deficient ( Sdf1 −/− ) platelets promotes alveolar regeneration in Thpo −/− mice a-c ) Platelets accumulate in the lungs of pneumonectomized Thpo −/− mice after intravascular infusion. Platelets were isolated from β-actin promoter driven- tdTomato ( ACTB-tdTomato ) mice and infused into Thpo −/− mice (a). Accumulation of tdTomato + CD41 + platelets in the lungs of Thpo −/− mice was determined by flow cytometry and immunostaining; Scale bar = 50 μm. d-g) Strategy to examine the influence of platelet-derived SDF1 on lung alveolar regeneration. Sdf1 +/+ and Sdf1 −/− platelets were isolated from wild type and Sdf1 Δ/Δ mice and infused into pneumonectomized Thpo −/− mice, respectively. Recovery of right lung volume (e), mass (f) and respiratory function (g) were determined in recipient mice. In panels (e) and (f), n =5 mice in all groups; P = 0.011 (e) and 0.026 (f) between mice transplanted with Sdf1 +/+ and Sdf1 −/− platelets. In panel (g), n = 6 mice ( Sdf1 +/+ ) and 4 mice ( Sdf1 −/− ) . P = 0.0013 (left) and 0.0042 (right) between two groups. h-k ) Expansion of AEC2s (h, j) and PCECs (i, k) in Thpo −/− mice after infusion of Sdf1 +/+ and Sdf1 −/− platelets. Representative immunostaining image of two transplanted groups and flow cytometry graph are shown; n = 5 mixw in all groups; P = 0.00011 (j) and 0.00081 (k) between two platelet types. Scale bar = 50 μm. l ) AEC1s in Thpo −/− mice following PNX and Sdf1 +/+ and Sdf1 −/− platelet transplantation. Scale bar = 50 μm. m-o ) Alveolar architecture of Thpo −/− mice receiving platelet infusion after PNX. Alveolar morphology was assessed by H E staining (m), and alveolar mean linear intercept (n) and number (o) were compared; n = 5 mice in both groups; P = 0.027 (n) and 0.0015 (o) between mice transplanted with Sdf1 +/+ and Sdf1 −/− platelets. Scale bar = 50 um. Error bar defines s.e.m., and line represents mean for panels e, f, g, j, k, n, o. Statistical difference between groups was assessed by unpaired two tail t-test.

    Article Snippet: To deplete circulating platelets, rat anti-mouse CD41 monoclonal antibody (BD Biosciences, clone MWReg30) was intraperitoneally injected into mice at dose of 10 mg/kg every three days.

    Techniques: Mouse Assay, Isolation, Flow Cytometry, Cytometry, Immunostaining, Derivative Assay, Transplantation Assay, Staining

    After left lung pneumonectomy (PNX), platelets are essential for the regrowth/regeneration of the remaining right lungs a, b ) After surgical removal of left lung lobe by PNX (a), surface expression of activation marker P-selectin on CD41 + platelets was examined by flow cytometry (b). Sham-operated mice underwent thoracotomy without lung resection. CD41 + P-selectin + activated platelets are denoted in yellow (b). c ) Weight (left) and volume (right) of the right lungs in wild type (WT) and thrombopoietin null ( Thpo −/− ) mice lacking circulating platelets. Left panel: n = 4 mice (both sham groups), n = 5 mice (WT group underwent PNX), and n = 4 mice ( Thpo −/− group with PNX). Right panel: n = 5 mice (WT with sham), n = 4 animals ( Thpo −/− with sham), and n = 5 animals for both PNX groups. d, e ) Lung inspiratory volume (d) and compliance (e) were restored in WT but not Thpo −/− mice at day 18 after PNX. n = 4 mice (both sham groups), n = 4 mice (WT with PNX), and n = 5 mice (pneumonectomized Thpo −/− group). f-j ) Bromodeoxyuridine (BrdU) incorporation in SFTPC+ type 2 alveolar epithelial cells (AEC2s) and VE-cadherin + . Panels (g) and (i): n = 4 mice in all tested groups; P = 0.00027 (g) and 0.0003 (i) between two groups. Scale bar = 50 μm. SFTPC, pro-surfactant protein C. j ) Distribution of type 1 alveolar epithelial cells (AEC1s) expressing aquaporin-5 and podoplanin in mice after PNX. k-q ) Lung regrowth and cell proliferation in thromobocytopenic mice after PNX. Mice were injected with anti-CD41 monoclonal antibody to deplete platelets (k). Propagation of AEC2s (l) and PCEC (m-n), localization of AEC1s (o), right lung volume (p) and arterial oxygen level (q) were measured in mice. Panels (l), (m): n = 4 mice in all groups; P = 0.00013 (l) and 0.00054 (m). Panel (p): n = 3 mice (IgG-treated sham), n = 5 mice (CD41 mAb-injected sham), and n = 5 mice (both PNX); Panel (q): n = 4 mice (both sham), n = 5 (PNX with IgG), and n = 4 (PNX with CD41 mAb). Scale bar = 50 μm. Error bar indicates stand error of mean (s.e.m.), and line represents mean for panels c, d, e, g, i, l, m, p, q. Difference between individual groups was compared by unpaired two-tail t-test between individual groups.

    Journal: Nature cell biology

    Article Title: Platelet-derived SDF1 primes pulmonary capillary vascular niche to drive lung alveolar regeneration

    doi: 10.1038/ncb3096

    Figure Lengend Snippet: After left lung pneumonectomy (PNX), platelets are essential for the regrowth/regeneration of the remaining right lungs a, b ) After surgical removal of left lung lobe by PNX (a), surface expression of activation marker P-selectin on CD41 + platelets was examined by flow cytometry (b). Sham-operated mice underwent thoracotomy without lung resection. CD41 + P-selectin + activated platelets are denoted in yellow (b). c ) Weight (left) and volume (right) of the right lungs in wild type (WT) and thrombopoietin null ( Thpo −/− ) mice lacking circulating platelets. Left panel: n = 4 mice (both sham groups), n = 5 mice (WT group underwent PNX), and n = 4 mice ( Thpo −/− group with PNX). Right panel: n = 5 mice (WT with sham), n = 4 animals ( Thpo −/− with sham), and n = 5 animals for both PNX groups. d, e ) Lung inspiratory volume (d) and compliance (e) were restored in WT but not Thpo −/− mice at day 18 after PNX. n = 4 mice (both sham groups), n = 4 mice (WT with PNX), and n = 5 mice (pneumonectomized Thpo −/− group). f-j ) Bromodeoxyuridine (BrdU) incorporation in SFTPC+ type 2 alveolar epithelial cells (AEC2s) and VE-cadherin + . Panels (g) and (i): n = 4 mice in all tested groups; P = 0.00027 (g) and 0.0003 (i) between two groups. Scale bar = 50 μm. SFTPC, pro-surfactant protein C. j ) Distribution of type 1 alveolar epithelial cells (AEC1s) expressing aquaporin-5 and podoplanin in mice after PNX. k-q ) Lung regrowth and cell proliferation in thromobocytopenic mice after PNX. Mice were injected with anti-CD41 monoclonal antibody to deplete platelets (k). Propagation of AEC2s (l) and PCEC (m-n), localization of AEC1s (o), right lung volume (p) and arterial oxygen level (q) were measured in mice. Panels (l), (m): n = 4 mice in all groups; P = 0.00013 (l) and 0.00054 (m). Panel (p): n = 3 mice (IgG-treated sham), n = 5 mice (CD41 mAb-injected sham), and n = 5 mice (both PNX); Panel (q): n = 4 mice (both sham), n = 5 (PNX with IgG), and n = 4 (PNX with CD41 mAb). Scale bar = 50 μm. Error bar indicates stand error of mean (s.e.m.), and line represents mean for panels c, d, e, g, i, l, m, p, q. Difference between individual groups was compared by unpaired two-tail t-test between individual groups.

    Article Snippet: To deplete circulating platelets, rat anti-mouse CD41 monoclonal antibody (BD Biosciences, clone MWReg30) was intraperitoneally injected into mice at dose of 10 mg/kg every three days.

    Techniques: Expressing, Activation Assay, Marker, Flow Cytometry, Cytometry, Mouse Assay, BrdU Incorporation Assay, Injection

    In mice lacking CCR2, the accumulation of Mφs and M2s in the colon postinfection is greatly reduced. CCR2 −/− and WT control mice (C57BL/6) were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of Mφs (F4/80 + cells) was conducted on sections of the proximal colon. Representative photographs of the F4/80 staining are shown in ( A ), and the quantitative analysis is shown in ( B ). Immunohistochemical staining of M2s (RELMα + cells) was also performed on sections of the proximal colon. Representative photographs of the RELMα staining are shown in ( C ), and the quantitative analysis is shown in ( D ). Scale bars, 100 μm. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Ly6G − Siglec-F − cells (as shown in E ). Representative plots of RELMα staining are shown in ( F ), and the data are shown graphically in ( G ). The values are the means ± SEM of five mice in each group. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

    doi: 10.4049/jimmunol.1400502

    Figure Lengend Snippet: In mice lacking CCR2, the accumulation of Mφs and M2s in the colon postinfection is greatly reduced. CCR2 −/− and WT control mice (C57BL/6) were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of Mφs (F4/80 + cells) was conducted on sections of the proximal colon. Representative photographs of the F4/80 staining are shown in ( A ), and the quantitative analysis is shown in ( B ). Immunohistochemical staining of M2s (RELMα + cells) was also performed on sections of the proximal colon. Representative photographs of the RELMα staining are shown in ( C ), and the quantitative analysis is shown in ( D ). Scale bars, 100 μm. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Ly6G − Siglec-F − cells (as shown in E ). Representative plots of RELMα staining are shown in ( F ), and the data are shown graphically in ( G ). The values are the means ± SEM of five mice in each group. * p

    Article Snippet: The cells were then stained with the following Abs to extracellular markers for 30 min on ice: PE rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 hamster, anti-mouse CD11c mAb (eBioscience), allophycocyanin–eFluor 780 rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), biotin rat, anti-mouse MHC class II (I-A/I-E) mAb (eBioscience) used in conjunction with PE-Vio770 mouse, anti-biotin mAb (Miltenyi Biotec, Bisley, U.K.), and VioGreen rat, anti-mouse CD45 mAb; or PE rat, anti-mouse CD103 mAb (BD Biosciences), PE rat, anti-mouse Siglec-F mAb (BD Biosciences), PE rat, anti-mouse Ly6G (BD Biosciences), FITC rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 rat, and anti-mouse CD45 mAb (eBioscience).

    Techniques: Mouse Assay, Infection, Immunohistochemistry, Staining, Isolation, Labeling, Flow Cytometry, Cytometry

    The proliferation of Mφs following infection with T. muris . AKR, C57BL/6, BALB/c, and CX3CR1 gfp/+ mice were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. In AKR, C57BL/6, and BALB/c mice, live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative plots of Ki-67 and BrdU staining are shown at selected time points postinfection ( A ). The data are shown at all time points in ( B ), where the values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. Ki-67 and BrdU staining in CX3CR1 gfp/+ mice was analyzed by gating on each of the four populations of monocytes and Mϕs (P1–P4, as defined in Fig. 5A ). Representative plots at selected time points postinfection are shown in ( C ). The gates were defined by staining with fluorochrome-labeled isotype control Abs in parallel (shown in Supplemental Fig. 3 ). The data are shown at all time points in ( D ) where the values are the means + SEM of five mice in each group, and the results are representative of two separate experiments. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

    doi: 10.4049/jimmunol.1400502

    Figure Lengend Snippet: The proliferation of Mφs following infection with T. muris . AKR, C57BL/6, BALB/c, and CX3CR1 gfp/+ mice were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. In AKR, C57BL/6, and BALB/c mice, live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative plots of Ki-67 and BrdU staining are shown at selected time points postinfection ( A ). The data are shown at all time points in ( B ), where the values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. Ki-67 and BrdU staining in CX3CR1 gfp/+ mice was analyzed by gating on each of the four populations of monocytes and Mϕs (P1–P4, as defined in Fig. 5A ). Representative plots at selected time points postinfection are shown in ( C ). The gates were defined by staining with fluorochrome-labeled isotype control Abs in parallel (shown in Supplemental Fig. 3 ). The data are shown at all time points in ( D ) where the values are the means + SEM of five mice in each group, and the results are representative of two separate experiments. * p

    Article Snippet: The cells were then stained with the following Abs to extracellular markers for 30 min on ice: PE rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 hamster, anti-mouse CD11c mAb (eBioscience), allophycocyanin–eFluor 780 rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), biotin rat, anti-mouse MHC class II (I-A/I-E) mAb (eBioscience) used in conjunction with PE-Vio770 mouse, anti-biotin mAb (Miltenyi Biotec, Bisley, U.K.), and VioGreen rat, anti-mouse CD45 mAb; or PE rat, anti-mouse CD103 mAb (BD Biosciences), PE rat, anti-mouse Siglec-F mAb (BD Biosciences), PE rat, anti-mouse Ly6G (BD Biosciences), FITC rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 rat, and anti-mouse CD45 mAb (eBioscience).

    Techniques: Infection, Mouse Assay, Injection, Isolation, Staining, Labeling, Flow Cytometry, Cytometry, BrdU Staining

    Flow cytometric analysis of lamina propria Mφs confirms the kinetics of M2 accumulation in the large intestine postinfection. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were either left uninfected or infected with a high level of T. muris ova. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells as shown in ( A ). Representative histogram plots of RELMα staining are shown in ( B ). Quantitative analysis of the staining is shown in ( C ). The values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

    doi: 10.4049/jimmunol.1400502

    Figure Lengend Snippet: Flow cytometric analysis of lamina propria Mφs confirms the kinetics of M2 accumulation in the large intestine postinfection. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were either left uninfected or infected with a high level of T. muris ova. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells as shown in ( A ). Representative histogram plots of RELMα staining are shown in ( B ). Quantitative analysis of the staining is shown in ( C ). The values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. * p

    Article Snippet: The cells were then stained with the following Abs to extracellular markers for 30 min on ice: PE rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 hamster, anti-mouse CD11c mAb (eBioscience), allophycocyanin–eFluor 780 rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), biotin rat, anti-mouse MHC class II (I-A/I-E) mAb (eBioscience) used in conjunction with PE-Vio770 mouse, anti-biotin mAb (Miltenyi Biotec, Bisley, U.K.), and VioGreen rat, anti-mouse CD45 mAb; or PE rat, anti-mouse CD103 mAb (BD Biosciences), PE rat, anti-mouse Siglec-F mAb (BD Biosciences), PE rat, anti-mouse Ly6G (BD Biosciences), FITC rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 rat, and anti-mouse CD45 mAb (eBioscience).

    Techniques: Flow Cytometry, Infection, Isolation, Staining, Labeling, Cytometry, Mouse Assay

    The vast majority of M2s do not proliferate. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative histogram plots of RELMα and BrdU staining are shown at selected time points postinfection ( A ). The RELMα + cells (M2s) were then analyzed for their BrdU content: the data are shown as the relative percentage of the BrdU + and BrdU − populations at all time points postinfection ( B ). The values are the means of five mice in each group, and the results are representative of two separate experiments.

    Journal: The Journal of Immunology Author Choice

    Article Title: Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

    doi: 10.4049/jimmunol.1400502

    Figure Lengend Snippet: The vast majority of M2s do not proliferate. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative histogram plots of RELMα and BrdU staining are shown at selected time points postinfection ( A ). The RELMα + cells (M2s) were then analyzed for their BrdU content: the data are shown as the relative percentage of the BrdU + and BrdU − populations at all time points postinfection ( B ). The values are the means of five mice in each group, and the results are representative of two separate experiments.

    Article Snippet: The cells were then stained with the following Abs to extracellular markers for 30 min on ice: PE rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 hamster, anti-mouse CD11c mAb (eBioscience), allophycocyanin–eFluor 780 rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), biotin rat, anti-mouse MHC class II (I-A/I-E) mAb (eBioscience) used in conjunction with PE-Vio770 mouse, anti-biotin mAb (Miltenyi Biotec, Bisley, U.K.), and VioGreen rat, anti-mouse CD45 mAb; or PE rat, anti-mouse CD103 mAb (BD Biosciences), PE rat, anti-mouse Siglec-F mAb (BD Biosciences), PE rat, anti-mouse Ly6G (BD Biosciences), FITC rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 rat, and anti-mouse CD45 mAb (eBioscience).

    Techniques: Infection, Injection, Isolation, Staining, Labeling, Flow Cytometry, Cytometry, BrdU Staining, Mouse Assay

    Cell viability and proliferation on different SF/P(LLA-CL) membranes. Notes: The first row of ( A ) shows the results of the live/dead kit test, and few dead cells can be seen. The second row of ( A ) shows Ki-67 protein staining, and the third row of ( A ) shows BrdU staining under laser scanning confocal microscopy. The scale bar indicates 50 μm in ( A ). The histogram of the positive percentage of Ki-67 protein staining is shown in ( B ). The histogram of the positive percentage of BrdU staining is shown in ( C ). * P

    Journal: International Journal of Nanomedicine

    Article Title: Electrospun nanofibrous SF/P(LLA-CL) membrane: a potential substratum for endothelial keratoplasty

    doi: 10.2147/IJN.S77706

    Figure Lengend Snippet: Cell viability and proliferation on different SF/P(LLA-CL) membranes. Notes: The first row of ( A ) shows the results of the live/dead kit test, and few dead cells can be seen. The second row of ( A ) shows Ki-67 protein staining, and the third row of ( A ) shows BrdU staining under laser scanning confocal microscopy. The scale bar indicates 50 μm in ( A ). The histogram of the positive percentage of Ki-67 protein staining is shown in ( B ). The histogram of the positive percentage of BrdU staining is shown in ( C ). * P

    Article Snippet: The cells were then subjected to mouse monoclonal anti-Ki-67 (1:200, BD Biosciences, Franklin Lakes, NJ, USA) at 4°C for 16 hours.

    Techniques: Staining, BrdU Staining, Confocal Microscopy

    PLC treatment reduces inflammation and endothelial dysfunction in rat TNBS-induced acute colitis. Representative microphotographs and morphometric evaluation of ICAM-1, VCAM-1, iNOS, CD31, PlGF, and BrDU staining of rat colon tissue from the different experimental groups: vehicle (50% ethanol, v/v), PLC (25 mg/kg intrarectal, twice daily for 1 week), TNBS and TNBS plus PLC (25 mg/kg intrarectal, twice daily for 1 week). Student's t -test: *, **, and *** P

    Journal: Clinical and Translational Gastroenterology

    Article Title: Propionyl-L-Carnitine is Efficacious in Ulcerative Colitis Through its Action on the Immune Function and Microvasculature

    doi: 10.1038/ctg.2014.4

    Figure Lengend Snippet: PLC treatment reduces inflammation and endothelial dysfunction in rat TNBS-induced acute colitis. Representative microphotographs and morphometric evaluation of ICAM-1, VCAM-1, iNOS, CD31, PlGF, and BrDU staining of rat colon tissue from the different experimental groups: vehicle (50% ethanol, v/v), PLC (25 mg/kg intrarectal, twice daily for 1 week), TNBS and TNBS plus PLC (25 mg/kg intrarectal, twice daily for 1 week). Student's t -test: *, **, and *** P

    Article Snippet: For rat tissues, mouse monoclonal anti-rat CD31 (BD Pharmingen, NJ, USA), was used.

    Techniques: Planar Chromatography, BrdU Staining

    Functional and structural analysis of the interactions between Sox9 and β-catenin. ( A ) Schematic representation of the β -catenin deletion mutants. ( B ) In vitro binding of β -catenin deletion mutants to 6xHis-tagged Sox9 bound to a nickel-resin. (I) Input; (B) bound to resin containing 6xHis-tagged Sox9. ( C ) Activation of TOPFLASH in C3H10T1/2 cells by Tcf3 is inhibited by Sox9 in a dose-dependent manner. ( D ) Sox9 inhibits binding of Tcf3 to β-catenin in vitro. Sox9 and 35 S-labeled Tcf3 proteins are incubated with 200 ng of 6xHis-tagged β-catenin immobilized to a nickel-resin. Of the input Tcf3 protein (lane 1 ) and bound Tcf3 proteins (lanes 2 - 4 ), 10% are resolved on SDS-PAGE. ( E ) Binding between Sox9 and β-catenin in Cos-7 cells. Cos-7 cells are cotransfected with 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9 for 24 h. Cell lysates are incubated with anti-β-catenin antibody and protein G-agarose beads under gentle agitation for 3 h at 4°C. The beads are washed three times with buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1% Triton X-100), resuspended in 10 μL of SDS-polyacrylamide gel electrophoresis sample loading buffer, and boiled for 2 min. Western immunoblotting is then performed. Anti-HA:HRP monoclonal antibody (Roche) and anti-myc:HRP antibody (Invitrogen) are diluted 1:1000 and 1:5000, respectively.

    Journal: Genes & Development

    Article Title: Interactions between Sox9 and ?-catenin control chondrocyte differentiation

    doi: 10.1101/gad.1171104

    Figure Lengend Snippet: Functional and structural analysis of the interactions between Sox9 and β-catenin. ( A ) Schematic representation of the β -catenin deletion mutants. ( B ) In vitro binding of β -catenin deletion mutants to 6xHis-tagged Sox9 bound to a nickel-resin. (I) Input; (B) bound to resin containing 6xHis-tagged Sox9. ( C ) Activation of TOPFLASH in C3H10T1/2 cells by Tcf3 is inhibited by Sox9 in a dose-dependent manner. ( D ) Sox9 inhibits binding of Tcf3 to β-catenin in vitro. Sox9 and 35 S-labeled Tcf3 proteins are incubated with 200 ng of 6xHis-tagged β-catenin immobilized to a nickel-resin. Of the input Tcf3 protein (lane 1 ) and bound Tcf3 proteins (lanes 2 - 4 ), 10% are resolved on SDS-PAGE. ( E ) Binding between Sox9 and β-catenin in Cos-7 cells. Cos-7 cells are cotransfected with 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9 for 24 h. Cell lysates are incubated with anti-β-catenin antibody and protein G-agarose beads under gentle agitation for 3 h at 4°C. The beads are washed three times with buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1% Triton X-100), resuspended in 10 μL of SDS-polyacrylamide gel electrophoresis sample loading buffer, and boiled for 2 min. Western immunoblotting is then performed. Anti-HA:HRP monoclonal antibody (Roche) and anti-myc:HRP antibody (Invitrogen) are diluted 1:1000 and 1:5000, respectively.

    Article Snippet: The following antibodies were used: goat polyclonal anti-Sox9 (1:100, Santa Cruz); rabbit polyclonal anti-Sox9 (1:100); rabbit polyclonal anti-HA (1:500, Covance); rabbit polyclonal anti-Cyclin D1 (1:100, Santa Cruz); and mouse monoclonal anti-β-catenin (1:100, BD Transduction Lab).

    Techniques: Functional Assay, In Vitro, Binding Assay, Activation Assay, Labeling, Incubation, SDS Page, Polyacrylamide Gel Electrophoresis, Western Blot

    ( A,B ) Sox9 inhibits β-catenin-mediated secondary-axis formation in Xenopus embryos. ( A ) Representative tail bud stage Xenopus embryos injected with the indicated RNAs into a single ventral-vegetal blastomere at the four-cell stage. ( B ) Summary of second axis assays from two separate experiments. Injection of mRNA encoding wild-type β-catenin (40 pg) results in a high frequency (77.8%) of embryos with secondary axes. Coinjection of increasing levels of Sox9 mRNA (0.5, 1.0, and 2.0 ng) with β -catenin (40 pg) results in a dose-dependent inhibition of β-catenin-induced secondary-axis formation. Sox9 (1-304) mRNA (2.0 ng) does not inhibit β-catenin-induced secondary-axis formation. Semiquantitative analysis of each embryo positive for a secondary axis (1 = weak, 2 = moderate, 3 = strong) further indicates that β-catenin-mediated secondary-axis formation is inhibited by increasing levels of Sox9, but not by Sox9 (1-304). ( C ) Proteasome inhibitor, MG132, restores the levels of β-catenin and Sox9 in 293 cells transfected with 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9. Mutant Sox9 (1-304) does not decrease the levels of β-catenin, and MG132 has no effect on the levels of either β-catenin or Sox9 in these experiments.

    Journal: Genes & Development

    Article Title: Interactions between Sox9 and ?-catenin control chondrocyte differentiation

    doi: 10.1101/gad.1171104

    Figure Lengend Snippet: ( A,B ) Sox9 inhibits β-catenin-mediated secondary-axis formation in Xenopus embryos. ( A ) Representative tail bud stage Xenopus embryos injected with the indicated RNAs into a single ventral-vegetal blastomere at the four-cell stage. ( B ) Summary of second axis assays from two separate experiments. Injection of mRNA encoding wild-type β-catenin (40 pg) results in a high frequency (77.8%) of embryos with secondary axes. Coinjection of increasing levels of Sox9 mRNA (0.5, 1.0, and 2.0 ng) with β -catenin (40 pg) results in a dose-dependent inhibition of β-catenin-induced secondary-axis formation. Sox9 (1-304) mRNA (2.0 ng) does not inhibit β-catenin-induced secondary-axis formation. Semiquantitative analysis of each embryo positive for a secondary axis (1 = weak, 2 = moderate, 3 = strong) further indicates that β-catenin-mediated secondary-axis formation is inhibited by increasing levels of Sox9, but not by Sox9 (1-304). ( C ) Proteasome inhibitor, MG132, restores the levels of β-catenin and Sox9 in 293 cells transfected with 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9. Mutant Sox9 (1-304) does not decrease the levels of β-catenin, and MG132 has no effect on the levels of either β-catenin or Sox9 in these experiments.

    Article Snippet: The following antibodies were used: goat polyclonal anti-Sox9 (1:100, Santa Cruz); rabbit polyclonal anti-Sox9 (1:100); rabbit polyclonal anti-HA (1:500, Covance); rabbit polyclonal anti-Cyclin D1 (1:100, Santa Cruz); and mouse monoclonal anti-β-catenin (1:100, BD Transduction Lab).

    Techniques: Injection, Inhibition, Transfection, Mutagenesis

    Functional and structural analysis of the interactions between Sox9 and β-catenin. ( A ) EMSAs performed using in vitro translated 3xHA-tagged Sox9 and 3xHA-tagged xTcf-3 proteins show that Sox9 does not bind to Tcf/Lef DNA-binding sites and that Tcf does not bind to Sox9 consensus DNA-binding sites. ( B ) Schematic representation of the Sox9 deletion mutants. ( C ) Activation of TOPFLASH by stβ-catenin is inhibited by Sox9 mutants containing the C-terminal transactivation domain. Cotransfection of 6x myc-tagged stβ-catenin and Sox9 mutants containing the C-terminal transactivation domain results in a reduction of the levels of these proteins. ( D ) In vitro binding of Sox9 deletion mutants to 6xHis-tagged β-catenin bound to a nickel-resin. (I) Input; (B) bound to resin containing 6xHis-tagged β-catenin.

    Journal: Genes & Development

    Article Title: Interactions between Sox9 and ?-catenin control chondrocyte differentiation

    doi: 10.1101/gad.1171104

    Figure Lengend Snippet: Functional and structural analysis of the interactions between Sox9 and β-catenin. ( A ) EMSAs performed using in vitro translated 3xHA-tagged Sox9 and 3xHA-tagged xTcf-3 proteins show that Sox9 does not bind to Tcf/Lef DNA-binding sites and that Tcf does not bind to Sox9 consensus DNA-binding sites. ( B ) Schematic representation of the Sox9 deletion mutants. ( C ) Activation of TOPFLASH by stβ-catenin is inhibited by Sox9 mutants containing the C-terminal transactivation domain. Cotransfection of 6x myc-tagged stβ-catenin and Sox9 mutants containing the C-terminal transactivation domain results in a reduction of the levels of these proteins. ( D ) In vitro binding of Sox9 deletion mutants to 6xHis-tagged β-catenin bound to a nickel-resin. (I) Input; (B) bound to resin containing 6xHis-tagged β-catenin.

    Article Snippet: The following antibodies were used: goat polyclonal anti-Sox9 (1:100, Santa Cruz); rabbit polyclonal anti-Sox9 (1:100); rabbit polyclonal anti-HA (1:500, Covance); rabbit polyclonal anti-Cyclin D1 (1:100, Santa Cruz); and mouse monoclonal anti-β-catenin (1:100, BD Transduction Lab).

    Techniques: Functional Assay, In Vitro, Binding Assay, Activation Assay, Cotransfection

    Model describing functional and physical interactions between Sox9 and β-catenin in chondrocytes. β-Catenin activates the Cyclin D1 gene and regulates chondrocyte proliferation. Sox9 activates ECM genes including Col2a1 and regulates chondrocyte differentiation. Sox9 inhibits β-catenin/Tcf-Lef activity by competing with the binding sites of Tcf/Lef within β-catenin. Sox9 also promotes degradation of β-catenin by the ubiquitination/26S proteasome pathway through formation of a Sox9:β-catenin complex. This results in inhibition of proliferation, and in delayed hypertrophic chondrocyte differentiation. In addition, formation of a Sox9:β-catenin complex also causes degradation of Sox9, inhibiting overt chondrocyte differentiation and accelerating hypertrophic chondrocyte differentiation. The model predicts that the relative levels of Sox9 and β-catenin control chondrocyte differentiation.

    Journal: Genes & Development

    Article Title: Interactions between Sox9 and ?-catenin control chondrocyte differentiation

    doi: 10.1101/gad.1171104

    Figure Lengend Snippet: Model describing functional and physical interactions between Sox9 and β-catenin in chondrocytes. β-Catenin activates the Cyclin D1 gene and regulates chondrocyte proliferation. Sox9 activates ECM genes including Col2a1 and regulates chondrocyte differentiation. Sox9 inhibits β-catenin/Tcf-Lef activity by competing with the binding sites of Tcf/Lef within β-catenin. Sox9 also promotes degradation of β-catenin by the ubiquitination/26S proteasome pathway through formation of a Sox9:β-catenin complex. This results in inhibition of proliferation, and in delayed hypertrophic chondrocyte differentiation. In addition, formation of a Sox9:β-catenin complex also causes degradation of Sox9, inhibiting overt chondrocyte differentiation and accelerating hypertrophic chondrocyte differentiation. The model predicts that the relative levels of Sox9 and β-catenin control chondrocyte differentiation.

    Article Snippet: The following antibodies were used: goat polyclonal anti-Sox9 (1:100, Santa Cruz); rabbit polyclonal anti-Sox9 (1:100); rabbit polyclonal anti-HA (1:500, Covance); rabbit polyclonal anti-Cyclin D1 (1:100, Santa Cruz); and mouse monoclonal anti-β-catenin (1:100, BD Transduction Lab).

    Techniques: Functional Assay, Activity Assay, Binding Assay, Inhibition

    Severe, generalized chondrodysplasia in β -catenin +/ flox(ex3) ; Col2a1-Cre embryos. ( A ) Gross appearance of E16.5 embryos. ( B ) Skeletons of E18.0 embryos stained by alcian blue followed by alizarin red. Mutant embryos are characterized by a very severe and generalized chondrodysplasia. ( C ) Gross appearance of E12.5 mutant embryos is comparable to that of wild-type littermates. Histological analysis of limb buds stained by hematoxylin and Treosin at 12.5 dpc. Both wild-type and mutant limb buds have discernible chondrogenic mesenchymal condensations. Immunohistochemistry shows comparable expression of Sox9 protein in condensed mesenchymal cells in E12.5 wild-type and mutant embryos. ( D ) Alcian blue and nuclear fast-red staining, PCNA staining, and immunohistochemistry of Sox9 protein in ulna of E16.5 wild-type and mutant embryos, respectively. Only cells surrounded by alcian blue-stainable matrix are PCNA-positive and express Sox9. Small round cells (arrows) lack expression of Sox9.

    Journal: Genes & Development

    Article Title: Interactions between Sox9 and ?-catenin control chondrocyte differentiation

    doi: 10.1101/gad.1171104

    Figure Lengend Snippet: Severe, generalized chondrodysplasia in β -catenin +/ flox(ex3) ; Col2a1-Cre embryos. ( A ) Gross appearance of E16.5 embryos. ( B ) Skeletons of E18.0 embryos stained by alcian blue followed by alizarin red. Mutant embryos are characterized by a very severe and generalized chondrodysplasia. ( C ) Gross appearance of E12.5 mutant embryos is comparable to that of wild-type littermates. Histological analysis of limb buds stained by hematoxylin and Treosin at 12.5 dpc. Both wild-type and mutant limb buds have discernible chondrogenic mesenchymal condensations. Immunohistochemistry shows comparable expression of Sox9 protein in condensed mesenchymal cells in E12.5 wild-type and mutant embryos. ( D ) Alcian blue and nuclear fast-red staining, PCNA staining, and immunohistochemistry of Sox9 protein in ulna of E16.5 wild-type and mutant embryos, respectively. Only cells surrounded by alcian blue-stainable matrix are PCNA-positive and express Sox9. Small round cells (arrows) lack expression of Sox9.

    Article Snippet: The following antibodies were used: goat polyclonal anti-Sox9 (1:100, Santa Cruz); rabbit polyclonal anti-Sox9 (1:100); rabbit polyclonal anti-HA (1:500, Covance); rabbit polyclonal anti-Cyclin D1 (1:100, Santa Cruz); and mouse monoclonal anti-β-catenin (1:100, BD Transduction Lab).

    Techniques: Staining, Mutagenesis, Immunohistochemistry, Expressing

    Analysis of skeletal phenotypes in conditional β -catenin -null mutants with the Col2a1-Cre transgene. ( A ) Gross appearance of newborn mice. ( B ) A cleft secondary palate in mutant newborn mice (the arrow). ( C,D ) Skeletons of newborn mice stained by alcian blue followed by alizarin red. ( E ) Alcian blue staining of the radius in E16.5 mice shows delay of endochondral bone formation in the mutant embryos. Staining by von Kossa's method shows no mineral deposition of the radius in E16.5 mutant embryos. ( F ) PCNA staining shows a decrease in PCNA-positive cells (brown nuclei) of the radius in E16.5 mutant embryos. The double arrows indicate the zone of proliferating chondrocytes. Boxed regions show a higher magnification of proliferating chondrocytes. BrdU incorporation also decreases in the radius in E16.5 mutant embryos. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t -test. ( * ) Statistically significant difference between wild-type and mutant embryos at p

    Journal: Genes & Development

    Article Title: Interactions between Sox9 and ?-catenin control chondrocyte differentiation

    doi: 10.1101/gad.1171104

    Figure Lengend Snippet: Analysis of skeletal phenotypes in conditional β -catenin -null mutants with the Col2a1-Cre transgene. ( A ) Gross appearance of newborn mice. ( B ) A cleft secondary palate in mutant newborn mice (the arrow). ( C,D ) Skeletons of newborn mice stained by alcian blue followed by alizarin red. ( E ) Alcian blue staining of the radius in E16.5 mice shows delay of endochondral bone formation in the mutant embryos. Staining by von Kossa's method shows no mineral deposition of the radius in E16.5 mutant embryos. ( F ) PCNA staining shows a decrease in PCNA-positive cells (brown nuclei) of the radius in E16.5 mutant embryos. The double arrows indicate the zone of proliferating chondrocytes. Boxed regions show a higher magnification of proliferating chondrocytes. BrdU incorporation also decreases in the radius in E16.5 mutant embryos. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t -test. ( * ) Statistically significant difference between wild-type and mutant embryos at p

    Article Snippet: The following antibodies were used: goat polyclonal anti-Sox9 (1:100, Santa Cruz); rabbit polyclonal anti-Sox9 (1:100); rabbit polyclonal anti-HA (1:500, Covance); rabbit polyclonal anti-Cyclin D1 (1:100, Santa Cruz); and mouse monoclonal anti-β-catenin (1:100, BD Transduction Lab).

    Techniques: Mouse Assay, Mutagenesis, Staining, BrdU Incorporation Assay

    Inhibition of cell proliferation and Cyclin D1 expression in Col2a1/Sox9 knock-in mutant embryos, and negative functional interactions between Sox9 and β-catenin in vitro. ( A ) PCNA staining shows a decrease in PCNA-positive cells (brown nuclei) in the radius of E16.5 mutant embryos. The double arrows indicate the zone of proliferating chondrocytes. Boxed regions show a higher magnification of proliferating chondrocytes. BrdU incorporation also decreases in the radius of E16.5 mutant embryos. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t -test. ( * ) Statistically significant difference between wild-type and mutant embryos at p

    Journal: Genes & Development

    Article Title: Interactions between Sox9 and ?-catenin control chondrocyte differentiation

    doi: 10.1101/gad.1171104

    Figure Lengend Snippet: Inhibition of cell proliferation and Cyclin D1 expression in Col2a1/Sox9 knock-in mutant embryos, and negative functional interactions between Sox9 and β-catenin in vitro. ( A ) PCNA staining shows a decrease in PCNA-positive cells (brown nuclei) in the radius of E16.5 mutant embryos. The double arrows indicate the zone of proliferating chondrocytes. Boxed regions show a higher magnification of proliferating chondrocytes. BrdU incorporation also decreases in the radius of E16.5 mutant embryos. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t -test. ( * ) Statistically significant difference between wild-type and mutant embryos at p

    Article Snippet: The following antibodies were used: goat polyclonal anti-Sox9 (1:100, Santa Cruz); rabbit polyclonal anti-Sox9 (1:100); rabbit polyclonal anti-HA (1:500, Covance); rabbit polyclonal anti-Cyclin D1 (1:100, Santa Cruz); and mouse monoclonal anti-β-catenin (1:100, BD Transduction Lab).

    Techniques: Inhibition, Expressing, Knock-In, Mutagenesis, Functional Assay, In Vitro, Staining, BrdU Incorporation Assay

    In mice lacking CCR2, the accumulation of Mφs and M2s in the colon postinfection is greatly reduced. CCR2 −/− and WT control mice (C57BL/6) were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of Mφs (F4/80 + cells) was conducted on sections of the proximal colon. Representative photographs of the F4/80 staining are shown in ( A ), and the quantitative analysis is shown in ( B ). Immunohistochemical staining of M2s (RELMα + cells) was also performed on sections of the proximal colon. Representative photographs of the RELMα staining are shown in ( C ), and the quantitative analysis is shown in ( D ). Scale bars, 100 μm. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Ly6G − Siglec-F − cells (as shown in E ). Representative plots of RELMα staining are shown in ( F ), and the data are shown graphically in ( G ). The values are the means ± SEM of five mice in each group. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

    doi: 10.4049/jimmunol.1400502

    Figure Lengend Snippet: In mice lacking CCR2, the accumulation of Mφs and M2s in the colon postinfection is greatly reduced. CCR2 −/− and WT control mice (C57BL/6) were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of Mφs (F4/80 + cells) was conducted on sections of the proximal colon. Representative photographs of the F4/80 staining are shown in ( A ), and the quantitative analysis is shown in ( B ). Immunohistochemical staining of M2s (RELMα + cells) was also performed on sections of the proximal colon. Representative photographs of the RELMα staining are shown in ( C ), and the quantitative analysis is shown in ( D ). Scale bars, 100 μm. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Ly6G − Siglec-F − cells (as shown in E ). Representative plots of RELMα staining are shown in ( F ), and the data are shown graphically in ( G ). The values are the means ± SEM of five mice in each group. * p

    Article Snippet: The cells were then stained with the following Abs to extracellular markers for 30 min on ice: PE rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 hamster, anti-mouse CD11c mAb (eBioscience), allophycocyanin–eFluor 780 rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), biotin rat, anti-mouse MHC class II (I-A/I-E) mAb (eBioscience) used in conjunction with PE-Vio770 mouse, anti-biotin mAb (Miltenyi Biotec, Bisley, U.K.), and VioGreen rat, anti-mouse CD45 mAb; or PE rat, anti-mouse CD103 mAb (BD Biosciences), PE rat, anti-mouse Siglec-F mAb (BD Biosciences), PE rat, anti-mouse Ly6G (BD Biosciences), FITC rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 rat, and anti-mouse CD45 mAb (eBioscience).

    Techniques: Mouse Assay, Infection, Immunohistochemistry, Staining, Isolation, Labeling, Flow Cytometry, Cytometry

    The proliferation of Mφs following infection with T. muris . AKR, C57BL/6, BALB/c, and CX3CR1 gfp/+ mice were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. In AKR, C57BL/6, and BALB/c mice, live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative plots of Ki-67 and BrdU staining are shown at selected time points postinfection ( A ). The data are shown at all time points in ( B ), where the values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. Ki-67 and BrdU staining in CX3CR1 gfp/+ mice was analyzed by gating on each of the four populations of monocytes and Mϕs (P1–P4, as defined in Fig. 5A ). Representative plots at selected time points postinfection are shown in ( C ). The gates were defined by staining with fluorochrome-labeled isotype control Abs in parallel (shown in Supplemental Fig. 3 ). The data are shown at all time points in ( D ) where the values are the means + SEM of five mice in each group, and the results are representative of two separate experiments. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

    doi: 10.4049/jimmunol.1400502

    Figure Lengend Snippet: The proliferation of Mφs following infection with T. muris . AKR, C57BL/6, BALB/c, and CX3CR1 gfp/+ mice were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. In AKR, C57BL/6, and BALB/c mice, live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative plots of Ki-67 and BrdU staining are shown at selected time points postinfection ( A ). The data are shown at all time points in ( B ), where the values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. Ki-67 and BrdU staining in CX3CR1 gfp/+ mice was analyzed by gating on each of the four populations of monocytes and Mϕs (P1–P4, as defined in Fig. 5A ). Representative plots at selected time points postinfection are shown in ( C ). The gates were defined by staining with fluorochrome-labeled isotype control Abs in parallel (shown in Supplemental Fig. 3 ). The data are shown at all time points in ( D ) where the values are the means + SEM of five mice in each group, and the results are representative of two separate experiments. * p

    Article Snippet: The cells were then stained with the following Abs to extracellular markers for 30 min on ice: PE rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 hamster, anti-mouse CD11c mAb (eBioscience), allophycocyanin–eFluor 780 rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), biotin rat, anti-mouse MHC class II (I-A/I-E) mAb (eBioscience) used in conjunction with PE-Vio770 mouse, anti-biotin mAb (Miltenyi Biotec, Bisley, U.K.), and VioGreen rat, anti-mouse CD45 mAb; or PE rat, anti-mouse CD103 mAb (BD Biosciences), PE rat, anti-mouse Siglec-F mAb (BD Biosciences), PE rat, anti-mouse Ly6G (BD Biosciences), FITC rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 rat, and anti-mouse CD45 mAb (eBioscience).

    Techniques: Infection, Mouse Assay, Injection, Isolation, Staining, Labeling, Flow Cytometry, Cytometry, BrdU Staining

    Flow cytometric analysis of lamina propria Mφs confirms the kinetics of M2 accumulation in the large intestine postinfection. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were either left uninfected or infected with a high level of T. muris ova. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells as shown in ( A ). Representative histogram plots of RELMα staining are shown in ( B ). Quantitative analysis of the staining is shown in ( C ). The values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

    doi: 10.4049/jimmunol.1400502

    Figure Lengend Snippet: Flow cytometric analysis of lamina propria Mφs confirms the kinetics of M2 accumulation in the large intestine postinfection. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were either left uninfected or infected with a high level of T. muris ova. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells as shown in ( A ). Representative histogram plots of RELMα staining are shown in ( B ). Quantitative analysis of the staining is shown in ( C ). The values are the means ± SEM of five mice in each group, and the results are representative of two separate experiments. * p

    Article Snippet: The cells were then stained with the following Abs to extracellular markers for 30 min on ice: PE rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 hamster, anti-mouse CD11c mAb (eBioscience), allophycocyanin–eFluor 780 rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), biotin rat, anti-mouse MHC class II (I-A/I-E) mAb (eBioscience) used in conjunction with PE-Vio770 mouse, anti-biotin mAb (Miltenyi Biotec, Bisley, U.K.), and VioGreen rat, anti-mouse CD45 mAb; or PE rat, anti-mouse CD103 mAb (BD Biosciences), PE rat, anti-mouse Siglec-F mAb (BD Biosciences), PE rat, anti-mouse Ly6G (BD Biosciences), FITC rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 rat, and anti-mouse CD45 mAb (eBioscience).

    Techniques: Flow Cytometry, Infection, Isolation, Staining, Labeling, Cytometry, Mouse Assay

    The vast majority of M2s do not proliferate. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative histogram plots of RELMα and BrdU staining are shown at selected time points postinfection ( A ). The RELMα + cells (M2s) were then analyzed for their BrdU content: the data are shown as the relative percentage of the BrdU + and BrdU − populations at all time points postinfection ( B ). The values are the means of five mice in each group, and the results are representative of two separate experiments.

    Journal: The Journal of Immunology Author Choice

    Article Title: Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

    doi: 10.4049/jimmunol.1400502

    Figure Lengend Snippet: The vast majority of M2s do not proliferate. Three different strains of mouse (AKR, C57BL/6, and BALB/c) were infected with a high level of T. muris ova. Each mouse was injected with 1.5 mg BrdU 4 h before it was killed. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mφs were analyzed by gating on viability stain–negative CD45 + CD11b + F4/80 + CD103 − Siglec-F − cells (as shown in Fig. 4A ). Representative histogram plots of RELMα and BrdU staining are shown at selected time points postinfection ( A ). The RELMα + cells (M2s) were then analyzed for their BrdU content: the data are shown as the relative percentage of the BrdU + and BrdU − populations at all time points postinfection ( B ). The values are the means of five mice in each group, and the results are representative of two separate experiments.

    Article Snippet: The cells were then stained with the following Abs to extracellular markers for 30 min on ice: PE rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 hamster, anti-mouse CD11c mAb (eBioscience), allophycocyanin–eFluor 780 rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), biotin rat, anti-mouse MHC class II (I-A/I-E) mAb (eBioscience) used in conjunction with PE-Vio770 mouse, anti-biotin mAb (Miltenyi Biotec, Bisley, U.K.), and VioGreen rat, anti-mouse CD45 mAb; or PE rat, anti-mouse CD103 mAb (BD Biosciences), PE rat, anti-mouse Siglec-F mAb (BD Biosciences), PE rat, anti-mouse Ly6G (BD Biosciences), FITC rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 rat, and anti-mouse CD45 mAb (eBioscience).

    Techniques: Infection, Injection, Isolation, Staining, Labeling, Flow Cytometry, Cytometry, BrdU Staining, Mouse Assay

    Effects of HIF inhibitors on HPF cell proliferation. HPFs were treated with the HIF-1α inhibitor KC7F2 or the HIF-2α inhibitor TC-S 7009 and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. ( A , B ) Cell proliferation as determined by BrdU assay. Cells were incubated with BrdU for 12 hrs. ( C , D ) Cell viability as determined by LDH assay. Data were expressed as a percentage of control (0 µM inhibitor) for each oxygen condition. The absorbance of control at normoxia for proliferation was 0.39 ± 0.06 (n = 3) and for hypoxia was 0.66 ± 0.16 (n = 3). The viability (%) was calculated as 100% -cytotoxicity %. Cytotoxicity % = [(LDH activity of sample -Spontaneous LDH activity)/(Maximum LDH activity -Spontaneous LDH activity)] × 100. The absorbance of control at normoxia for LDH activity was 0.17 (n = 2) and for hypoxia was 0.16 (n = 2). Maximum and spontaneous LDH activity values for normoxia were 0.26 and 0.17 (n = 2), respectively. Maximum and spontaneous LDH activity values for hypoxia were 0.27 and 0.17 (n = 2), respectively. Values represent means ± SE for proliferation and means for viability.

    Journal: Scientific Reports

    Article Title: Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling

    doi: 10.1038/s41598-018-21073-x

    Figure Lengend Snippet: Effects of HIF inhibitors on HPF cell proliferation. HPFs were treated with the HIF-1α inhibitor KC7F2 or the HIF-2α inhibitor TC-S 7009 and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. ( A , B ) Cell proliferation as determined by BrdU assay. Cells were incubated with BrdU for 12 hrs. ( C , D ) Cell viability as determined by LDH assay. Data were expressed as a percentage of control (0 µM inhibitor) for each oxygen condition. The absorbance of control at normoxia for proliferation was 0.39 ± 0.06 (n = 3) and for hypoxia was 0.66 ± 0.16 (n = 3). The viability (%) was calculated as 100% -cytotoxicity %. Cytotoxicity % = [(LDH activity of sample -Spontaneous LDH activity)/(Maximum LDH activity -Spontaneous LDH activity)] × 100. The absorbance of control at normoxia for LDH activity was 0.17 (n = 2) and for hypoxia was 0.16 (n = 2). Maximum and spontaneous LDH activity values for normoxia were 0.26 and 0.17 (n = 2), respectively. Maximum and spontaneous LDH activity values for hypoxia were 0.27 and 0.17 (n = 2), respectively. Values represent means ± SE for proliferation and means for viability.

    Article Snippet: The following antibodies were added, and membranes were incubated at 4 °C overnight: polyclonal rabbit anti-NFATc2 (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, Cat. No: 13034), monoclonal mouse anti-cyclin A2 (1:1000 dilution, Cell signaling, Beverly, MA, Cat. No: 4656), monoclonal mouse anti-cyclin D1 (1:750 dilution, Santa Cruz Biotechnology, Cat. No: 8396), monoclonal mouse anti-cyclin E1 (1:1000 dilution, Cell signaling, Cat. No: 4129), polyclonal rabbit anti-CDK2 (1:750 dilution, Santa Cruz Biotechnology, Cat. No: 163), polyclonal rabbit anti-CDK4 (1:750 dilution, Santa Cruz Biotechnology, Cat. No: 260), polyclonal rabbit anti-CDK6 (1:750 dilution, Santa Cruz Biotechnology, Cat. No: 177), monoclonal mouse anti-HIF-1α (1:300 dilution, BD biosciences, La Jolla, CA, Cat. No: 610958), polyclonal rabbit anti-HIF-2α (1:500 dilution, Novus biologicals, Littleton, CO, Cat. No: 100-122), and monoclonal mouse anti-actin (β-actin) (1:2000 -1:5000 dilutions, Thermo Fisher Scientific, Waltham, MA, Cat. No: MA5-15739).

    Techniques: BrdU Staining, Incubation, Lactate Dehydrogenase Assay, Activity Assay

    Inhibition and silencing of HIF-2α reduces hypoxia-mediated NFATc2 nuclear translocation. ( A ) Immunofluorescence staining of NFATc2 in HPFs treated with HIF-1α and HIF-2α inhibitors (KC7F2, 10 µM and TC-S 7009, 50 µM, respectively) and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. Scale Bar: 50 µm. ( B ) Percentages of NFATc2 nuclear-translocated cells were determined by counting the cells with NFATc2 nuclear localization signals compared to the total number of cells. ( C ) Lentiviral infection efficiency. BC = Blank control, VC = Vector control. ( D ) Western blot showing HIF-1α and HIF-2α silencing efficiency. ( E ) Quantitative representation of protein expression of HIF-1α and HIF-2α protein expression with HIF silencing. ( F ) Immunofluorescence staining of NFATc2 in HPFs treated with shRNA lentiviral constructs (MOI 100) of HIF-1α and HIF-2α and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. Scale Bar: 50 µm. ( G ) Percentages of NFATc2 nuclear-translocated cells were determined by counting the cells with NFATc2 nuclear localization signals compared to the total number of cells. ( H ) HEK 293Ts were co-transfected with an NFAT reporter plasmid and HIF-1α or HIF-2α expression vector for 24 hrs. The reporter activities were measured as the ratio of Firefly/Renilla luciferase activities. Data represent means ± SE. *p

    Journal: Scientific Reports

    Article Title: Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling

    doi: 10.1038/s41598-018-21073-x

    Figure Lengend Snippet: Inhibition and silencing of HIF-2α reduces hypoxia-mediated NFATc2 nuclear translocation. ( A ) Immunofluorescence staining of NFATc2 in HPFs treated with HIF-1α and HIF-2α inhibitors (KC7F2, 10 µM and TC-S 7009, 50 µM, respectively) and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. Scale Bar: 50 µm. ( B ) Percentages of NFATc2 nuclear-translocated cells were determined by counting the cells with NFATc2 nuclear localization signals compared to the total number of cells. ( C ) Lentiviral infection efficiency. BC = Blank control, VC = Vector control. ( D ) Western blot showing HIF-1α and HIF-2α silencing efficiency. ( E ) Quantitative representation of protein expression of HIF-1α and HIF-2α protein expression with HIF silencing. ( F ) Immunofluorescence staining of NFATc2 in HPFs treated with shRNA lentiviral constructs (MOI 100) of HIF-1α and HIF-2α and exposed to normoxia and hypoxia (1% O 2 ) for 3 days. Scale Bar: 50 µm. ( G ) Percentages of NFATc2 nuclear-translocated cells were determined by counting the cells with NFATc2 nuclear localization signals compared to the total number of cells. ( H ) HEK 293Ts were co-transfected with an NFAT reporter plasmid and HIF-1α or HIF-2α expression vector for 24 hrs. The reporter activities were measured as the ratio of Firefly/Renilla luciferase activities. Data represent means ± SE. *p

    Article Snippet: The following antibodies were added, and membranes were incubated at 4 °C overnight: polyclonal rabbit anti-NFATc2 (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, Cat. No: 13034), monoclonal mouse anti-cyclin A2 (1:1000 dilution, Cell signaling, Beverly, MA, Cat. No: 4656), monoclonal mouse anti-cyclin D1 (1:750 dilution, Santa Cruz Biotechnology, Cat. No: 8396), monoclonal mouse anti-cyclin E1 (1:1000 dilution, Cell signaling, Cat. No: 4129), polyclonal rabbit anti-CDK2 (1:750 dilution, Santa Cruz Biotechnology, Cat. No: 163), polyclonal rabbit anti-CDK4 (1:750 dilution, Santa Cruz Biotechnology, Cat. No: 260), polyclonal rabbit anti-CDK6 (1:750 dilution, Santa Cruz Biotechnology, Cat. No: 177), monoclonal mouse anti-HIF-1α (1:300 dilution, BD biosciences, La Jolla, CA, Cat. No: 610958), polyclonal rabbit anti-HIF-2α (1:500 dilution, Novus biologicals, Littleton, CO, Cat. No: 100-122), and monoclonal mouse anti-actin (β-actin) (1:2000 -1:5000 dilutions, Thermo Fisher Scientific, Waltham, MA, Cat. No: MA5-15739).

    Techniques: Inhibition, Translocation Assay, Immunofluorescence, Staining, Infection, Plasmid Preparation, Western Blot, Expressing, shRNA, Construct, Transfection, Luciferase