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Becton Dickinson mouse monoclonal anti brdu antibody
Effect of acute ACTH treatment on <t>BrdU-pulse-chase-labelled</t> cells in the adult mouse adrenal cortex. Adrenal sections from female F1 mice treated acutely with ACTH 6 weeks after 1-week BrdU infusion, <t>immunostained</t> for (A,B) BrdU, (C,D) Ki67 and (E,F) BrdU/Ki67. Mice (n =6/group) were injected with saline vehicle (A,C,E) or ACTH (B,D,F), as described in the Materials and Methods section. Ki67-positive cells are indicated by brown nuclear staining while BrdU-labelled cells are identified by blue nuclear staining. Typical examples of BrdU/Ki67 dual-labelled cells are shown by arrows in F. Sections C and D were lightly counterstained with haematoxylin but sections A,B,E F were mounted without counterstaining. Scale = 100 µm.
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Images

1) Product Images from "Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex"

Article Title: Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081865

Effect of acute ACTH treatment on BrdU-pulse-chase-labelled cells in the adult mouse adrenal cortex. Adrenal sections from female F1 mice treated acutely with ACTH 6 weeks after 1-week BrdU infusion, immunostained for (A,B) BrdU, (C,D) Ki67 and (E,F) BrdU/Ki67. Mice (n =6/group) were injected with saline vehicle (A,C,E) or ACTH (B,D,F), as described in the Materials and Methods section. Ki67-positive cells are indicated by brown nuclear staining while BrdU-labelled cells are identified by blue nuclear staining. Typical examples of BrdU/Ki67 dual-labelled cells are shown by arrows in F. Sections C and D were lightly counterstained with haematoxylin but sections A,B,E F were mounted without counterstaining. Scale = 100 µm.
Figure Legend Snippet: Effect of acute ACTH treatment on BrdU-pulse-chase-labelled cells in the adult mouse adrenal cortex. Adrenal sections from female F1 mice treated acutely with ACTH 6 weeks after 1-week BrdU infusion, immunostained for (A,B) BrdU, (C,D) Ki67 and (E,F) BrdU/Ki67. Mice (n =6/group) were injected with saline vehicle (A,C,E) or ACTH (B,D,F), as described in the Materials and Methods section. Ki67-positive cells are indicated by brown nuclear staining while BrdU-labelled cells are identified by blue nuclear staining. Typical examples of BrdU/Ki67 dual-labelled cells are shown by arrows in F. Sections C and D were lightly counterstained with haematoxylin but sections A,B,E F were mounted without counterstaining. Scale = 100 µm.

Techniques Used: Pulse Chase, Mouse Assay, Injection, Staining

Effect of acute ACTH treatment on BrdU pulse-chase-labelled cell numbers in the adult mouse adrenal cortex. BrdU and Ki67-labelled cell numbers in control and 4 h ACTH treatment groups (n=6) of female F1 mice, treated 6 weeks after 1-week BrdU infusion. Mean percentage (±SEM) of total cell numbers are shown for each zone and treatment that were (A) BrdU-positive/Ki67-negative, (B) BrdU-negative/Ki67-positive, (C) BrdU-positive/Ki67-positive (dual-labelled) and (D) BrdU-negative/Ki67-negative (unlabelled). Cells were counted in ZG, outer ZF, inner ZF and ZR in dual-BrdU/Ki67-immunostained left adrenal sections, as described in the Materials and Methods section.
Figure Legend Snippet: Effect of acute ACTH treatment on BrdU pulse-chase-labelled cell numbers in the adult mouse adrenal cortex. BrdU and Ki67-labelled cell numbers in control and 4 h ACTH treatment groups (n=6) of female F1 mice, treated 6 weeks after 1-week BrdU infusion. Mean percentage (±SEM) of total cell numbers are shown for each zone and treatment that were (A) BrdU-positive/Ki67-negative, (B) BrdU-negative/Ki67-positive, (C) BrdU-positive/Ki67-positive (dual-labelled) and (D) BrdU-negative/Ki67-negative (unlabelled). Cells were counted in ZG, outer ZF, inner ZF and ZR in dual-BrdU/Ki67-immunostained left adrenal sections, as described in the Materials and Methods section.

Techniques Used: Pulse Chase, Mouse Assay

2) Product Images from "BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior"

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

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

doi: 10.1523/JNEUROSCI.0550-10.2010

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).
Figure Legend 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).

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

3) Product Images from "Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design"

Article Title: Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1000355

Effects of Spadin on neurogenesis and CREB activation. (A–B) Spadin increased neurogenesis (A). Top, representative photomicrographs of BrdU-labeled neurons in the dentate gyrus of the mouse hippocampus treated either with saline or with spadin (i.v., 10 −6 M) for 4 d. Bottom, double labeling of BrdU-labeled neurons either with GFAP (glial marker) or with DCX (neuronal precursor marker), showing a co-localization only with DCX, and not with GFAP. (B) Quantitation of BrdU positive cells of hippocampus treated with saline, fluoxetine, or spadin (10 −5 M) for 4 d. 85% of BrdU-labeled cells were positive to DCX. Data are number of BrdU + or DCX + cells in mouse hippocampus ( n = 5) (F 2,53 = 35.27; *** p
Figure Legend Snippet: Effects of Spadin on neurogenesis and CREB activation. (A–B) Spadin increased neurogenesis (A). Top, representative photomicrographs of BrdU-labeled neurons in the dentate gyrus of the mouse hippocampus treated either with saline or with spadin (i.v., 10 −6 M) for 4 d. Bottom, double labeling of BrdU-labeled neurons either with GFAP (glial marker) or with DCX (neuronal precursor marker), showing a co-localization only with DCX, and not with GFAP. (B) Quantitation of BrdU positive cells of hippocampus treated with saline, fluoxetine, or spadin (10 −5 M) for 4 d. 85% of BrdU-labeled cells were positive to DCX. Data are number of BrdU + or DCX + cells in mouse hippocampus ( n = 5) (F 2,53 = 35.27; *** p

Techniques Used: Activation Assay, Labeling, Marker, Quantitation Assay

4) Product Images from "Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior"

Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00863

Neurogenesis in Sort1 −/− mice. (A) BrdU staining of dentate gyrus slices from WT and Sort1 −/− mice treated with saline or spadin for 4 days. (B) Double labeling of BrdU-labeled neurons with DCX (neuronal precursor marker), showing a colocalization in magnificated regions, bar: 50 μm. (C) 4 Days spadin treatment increased the number of BrdU cells in hippocampus from WT and Sort1 −/− mice ( n = 5 per genotype), expressed as mean ± SEM. ∗∗∗ p
Figure Legend Snippet: Neurogenesis in Sort1 −/− mice. (A) BrdU staining of dentate gyrus slices from WT and Sort1 −/− mice treated with saline or spadin for 4 days. (B) Double labeling of BrdU-labeled neurons with DCX (neuronal precursor marker), showing a colocalization in magnificated regions, bar: 50 μm. (C) 4 Days spadin treatment increased the number of BrdU cells in hippocampus from WT and Sort1 −/− mice ( n = 5 per genotype), expressed as mean ± SEM. ∗∗∗ p

Techniques Used: Mouse Assay, BrdU Staining, Labeling, Marker

5) Product Images from "EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele"

Article Title: EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv691

EXO1 influences DNA repair and DNA replication in Nbs1 mutants. ( A ) Schematic illustration of the SA-GFP based SSA assay. ( B) Western blotting of cells transfected with or without a vector expressing human NBS1. The genotypes are abbreviated throughout the figure as follows: wild type = W , Nbs1 ΔB/ΔB = N , Exo1 −/− = E , and Nbs1 ΔB/ΔB Exo −/− = NE . ( C ) Quantification of SSA mediated repair plotted as the percentage of GFP positive cells. Values for NE are corrected for the reduced percentage of cells in S/G2 determined by BrdU and PI staining (mean 58% compared to a mean of 73% in the other genotypes). The fold rescue with NBS1 expression is the same in N and NE (2.2 fold). ( D ) Measurement of replication tract lengths following CldU or IdU pulse labeling. ( E ) Calculation of replication fork velocity in the indicated genotypes (as described in ‘Materials and Methods’ section). Examples of representative forks from W and NE cultures are shown for comparison. ( F ) Assessment of replication fork restart following 1 mM HU treatment using the indicated scheme. Relative tract ratio is calculated by dividing the length of the IdU tract by that of the CldU tract ( = 2 under unperturbed conditions). Thus, higher values indicate faster restart following HU removal.
Figure Legend Snippet: EXO1 influences DNA repair and DNA replication in Nbs1 mutants. ( A ) Schematic illustration of the SA-GFP based SSA assay. ( B) Western blotting of cells transfected with or without a vector expressing human NBS1. The genotypes are abbreviated throughout the figure as follows: wild type = W , Nbs1 ΔB/ΔB = N , Exo1 −/− = E , and Nbs1 ΔB/ΔB Exo −/− = NE . ( C ) Quantification of SSA mediated repair plotted as the percentage of GFP positive cells. Values for NE are corrected for the reduced percentage of cells in S/G2 determined by BrdU and PI staining (mean 58% compared to a mean of 73% in the other genotypes). The fold rescue with NBS1 expression is the same in N and NE (2.2 fold). ( D ) Measurement of replication tract lengths following CldU or IdU pulse labeling. ( E ) Calculation of replication fork velocity in the indicated genotypes (as described in ‘Materials and Methods’ section). Examples of representative forks from W and NE cultures are shown for comparison. ( F ) Assessment of replication fork restart following 1 mM HU treatment using the indicated scheme. Relative tract ratio is calculated by dividing the length of the IdU tract by that of the CldU tract ( = 2 under unperturbed conditions). Thus, higher values indicate faster restart following HU removal.

Techniques Used: SSA Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Staining, Labeling

6) Product Images from "PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA"

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

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200806068

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.
Figure Legend 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.

Techniques Used: Immunofluorescence, Labeling

7) Product Images from "SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis"

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

Journal: Genes & Development

doi: 10.1101/gad.1412706

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.
Figure Legend 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.

Techniques Used: Staining, Immunofluorescence, Incubation, Marker, FACS

8) Product Images from "Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells"

Article Title: Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells

Journal: Stem Cells and Development

doi: 10.1089/scd.2010.0159

Sustained expression of RAD51 protein throughout the cell cycle in mESC. Exponentially growing mESCs and fibroblasts (FIB) were pulsed with BrdU, incubated with RAD51 antibody and 7-amino-actinomycin D (7AAD) (DNA content marker), and analyzed by FACS
Figure Legend Snippet: Sustained expression of RAD51 protein throughout the cell cycle in mESC. Exponentially growing mESCs and fibroblasts (FIB) were pulsed with BrdU, incubated with RAD51 antibody and 7-amino-actinomycin D (7AAD) (DNA content marker), and analyzed by FACS

Techniques Used: Expressing, Incubation, Marker, FACS

9) Product Images from "Replication stress induced site-specific phosphorylation targets WRN to the ubiquitin-proteasome pathway"

Article Title: Replication stress induced site-specific phosphorylation targets WRN to the ubiquitin-proteasome pathway

Journal: Oncotarget

doi:

ATR-mediated WRN phosphorylation is critical for replication fork processes upon replication stress A. WRN is recruited to replication forks sites independently of its S1141 phosphorylation. WS cells stably expressing EGFP-tagged WT or EGFP-tagged S1141A WRN were pulse-labeled with 50 μM EdU for 90 min and then treated with 1 μM CPT for 1 h. After 8 h, cells were fixed with 4% paraformaldehyde and immunostained with anti-RPA2 antibodies. Subsequently, the Click-IT reaction was carried out to detect EdU signal. Representative three-dimensional deconvoluted confocal images are shown. Scale bars are 5 μm. B. Replication fork progression is inefficient in WS+S1141A cells in response to replication stress. DNA fiber length distributions in WS+WT, WS, and WS+S1141A cells are illustrated before and after CPT treatment. Cells were labeled with IdU for 30 min, treated with ± CPT (1 μM) for 60 min, and labeled with CIdU for another 30 min. DNA fibers were immunostained with anti-BrdU (rat and mouse) antibodies, images were captured using fluorescence microscopy, and IdU (mock-treated) and CldU (CPT-treated) tract lengths were measured using Axiovison Software. More than 200 DNA fibers were evaluated in each sample. Each data point is the average of three independent experiments. Error bars represent STDEV. C. - E. WRN phosphorylation suppresses new origin firing and replication fork stalling in response to replication stress. C. Percentages of replication fork restarts in CPT-treated WS+WT, WS, and WS+S1141A cells relative to mock-treated cells are shown; levels were evaluated using the following formula: (IdU→CldU)/[IdU+(IdU→CldU)]. D. The graph shows fold changes in new origin firing in CPT-treated WS+WT, WS, and WS+S1141A cells relative to mock-treated cells calculated using the formula: CldU/[CldU+(IdU→CldU)]. E. The graph shows fold changes in replication forks stalling in CPT-treated WS+WT, WS, and WS+S1141A cells relative to mock-treated cells evaluated using the formula: IdU/[IdU+(IdU→CldU)]. More than 100 DNA fibers in each sample were evaluated. Each data point is the average of three independent experiments. Error bars represent STDEV. * indicates P
Figure Legend Snippet: ATR-mediated WRN phosphorylation is critical for replication fork processes upon replication stress A. WRN is recruited to replication forks sites independently of its S1141 phosphorylation. WS cells stably expressing EGFP-tagged WT or EGFP-tagged S1141A WRN were pulse-labeled with 50 μM EdU for 90 min and then treated with 1 μM CPT for 1 h. After 8 h, cells were fixed with 4% paraformaldehyde and immunostained with anti-RPA2 antibodies. Subsequently, the Click-IT reaction was carried out to detect EdU signal. Representative three-dimensional deconvoluted confocal images are shown. Scale bars are 5 μm. B. Replication fork progression is inefficient in WS+S1141A cells in response to replication stress. DNA fiber length distributions in WS+WT, WS, and WS+S1141A cells are illustrated before and after CPT treatment. Cells were labeled with IdU for 30 min, treated with ± CPT (1 μM) for 60 min, and labeled with CIdU for another 30 min. DNA fibers were immunostained with anti-BrdU (rat and mouse) antibodies, images were captured using fluorescence microscopy, and IdU (mock-treated) and CldU (CPT-treated) tract lengths were measured using Axiovison Software. More than 200 DNA fibers were evaluated in each sample. Each data point is the average of three independent experiments. Error bars represent STDEV. C. - E. WRN phosphorylation suppresses new origin firing and replication fork stalling in response to replication stress. C. Percentages of replication fork restarts in CPT-treated WS+WT, WS, and WS+S1141A cells relative to mock-treated cells are shown; levels were evaluated using the following formula: (IdU→CldU)/[IdU+(IdU→CldU)]. D. The graph shows fold changes in new origin firing in CPT-treated WS+WT, WS, and WS+S1141A cells relative to mock-treated cells calculated using the formula: CldU/[CldU+(IdU→CldU)]. E. The graph shows fold changes in replication forks stalling in CPT-treated WS+WT, WS, and WS+S1141A cells relative to mock-treated cells evaluated using the formula: IdU/[IdU+(IdU→CldU)]. More than 100 DNA fibers in each sample were evaluated. Each data point is the average of three independent experiments. Error bars represent STDEV. * indicates P

Techniques Used: Stable Transfection, Expressing, Labeling, Cycling Probe Technology, Fluorescence, Microscopy, Software

10) Product Images from "Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior"

Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00863

Neurogenesis in Sort1 −/− mice. (A) BrdU staining of dentate gyrus slices from WT and Sort1 −/− mice treated with saline or spadin for 4 days. (B) Double labeling of BrdU-labeled neurons with DCX (neuronal precursor marker), showing a colocalization in magnificated regions, bar: 50 μm. (C) 4 Days spadin treatment increased the number of BrdU cells in hippocampus from WT and Sort1 −/− mice ( n = 5 per genotype), expressed as mean ± SEM. ∗∗∗ p
Figure Legend Snippet: Neurogenesis in Sort1 −/− mice. (A) BrdU staining of dentate gyrus slices from WT and Sort1 −/− mice treated with saline or spadin for 4 days. (B) Double labeling of BrdU-labeled neurons with DCX (neuronal precursor marker), showing a colocalization in magnificated regions, bar: 50 μm. (C) 4 Days spadin treatment increased the number of BrdU cells in hippocampus from WT and Sort1 −/− mice ( n = 5 per genotype), expressed as mean ± SEM. ∗∗∗ p

Techniques Used: Mouse Assay, BrdU Staining, Labeling, Marker

Related Articles

Amplification:

Article Title: Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
Article Snippet: After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA. .. Isolated early and late replicating DNA were amplified by whole-genome amplification (WGA) (Sigma-Aldrich, St Louis, MO, USA, GenomePlex), labeled with Cy3 and Cy5, and hybridized to a mouse whole-genome microarray (NimbleGen Symtems, Madison WIS, USA, 2006-07-26_MM8_WG_CGH or 100718_MM9_WG_CGH_HX3).

Whole Genome Amplification:

Article Title: Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
Article Snippet: After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA. .. Isolated early and late replicating DNA were amplified by whole-genome amplification (WGA) (Sigma-Aldrich, St Louis, MO, USA, GenomePlex), labeled with Cy3 and Cy5, and hybridized to a mouse whole-genome microarray (NimbleGen Symtems, Madison WIS, USA, 2006-07-26_MM8_WG_CGH or 100718_MM9_WG_CGH_HX3).

Cycling Probe Technology:

Article Title: PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA
Article Snippet: 2 × 106 m5S and HeLa cells were pulse labeled for 20 min with 100 μM IdU, washed with PBS twice, and pulse labeled for 20 min with 100 μM CldU with or without 1 μM CPT. .. 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.

Cytometry:

Article Title: Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells
Article Snippet: After washing, anti-rabbit phycoerythrin 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 7-amino-actinomycin D (7AAD) fluorescent dye (BD Biosciences). .. Data acquisition and analysis were performed using both a flow cytometer (cell counter FC500) and software (XL-MCL) from Beckman Coulter.

Article Title: SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis
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). .. Data acquisition and analysis were performed using both flow cytometer (cell counter FC500) and software (XL-MCL) from Beckman Coulter.

Article Title: Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
Article Snippet: These cells were resuspended in PBS containing 1% FBS, stained with propidium iodide (50 μg/ml) for 30 min in the presence of RNaseA (0.5 mg/ml), and then were sorted into early and late S phase fractions by flow cytometry. .. After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA.

Blocking Assay:

Article Title: EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele
Article Snippet: .. For immunodetection, slides were blocked for 1 h in blocking solution (PBS, 1% BSA, 0.1% Tween20) and incubated overnight at 4°C with a mix of rat anti-BrdU monoclonal antibody (1:500 in blocking solution, AbD Serotec) that recognizes CldU, and mouse anti-BrdU monoclonal antibody (1:500 in blocking solution, Becton Dickinson) that recognizes IdU. .. Slides were washed three times with PBS and fixed with 4% formaldehyde for 10 min, following by three times washing with PBS and three times with blocking solution.

Article Title: PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA
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. .. All antibodies were diluted in blocking solution (1% [wt/vol] blocking reagent in PBS and 0.05% Tween 20; Roche).

Article Title: Muscle regeneration is undisturbed by repeated polytraumatic injury
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. .. An Alexa Fluor 488 anti-mouse secondary antibody and Alexa Fluor 546 anti-rabbit antibody (Molecular Probes, #A11001, #A11010) were used to visualize the labeling at 1:200 dilution and Hoechst nuclear staining (Invitrogen, #33342) at 1:1,000 dilution was used to label the nuclei of the cells.

Article Title: Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex
Article Snippet: .. After blocking in 20% normal rabbit serum in TBS for 30 min, sections were immunostained using a mouse monoclonal anti BrdU antibody (Becton Dickinson, 347580; 1:200), followed by a biotinylated rabbit anti-mouse second antibody (Dako, E0354; 1:200) and then avidin-biotin horseradish peroxidase (HRP) (Dako, K0355; 1:1000) using the 3,3′-Diaminobenzidine (DAB) isopac system as substrate (Sigma, D9015). .. BrdU/Ki67 dual-immunostaining was carried out after dewaxing, blocking and HIER as described above, using a sheep polyclonal anti-BrdU antibody (Fitzgerald Industries, 20-BS17; 1:7000), followed by a biotinylated rabbit anti-sheep second antibody (Vector Labs, BA-6000; 1:500) and streptavidin-alkaline phosphatase (Dako, D0396; 1:1000) using Fast Blue (Sigma, F3378; 1mg/ml in 0.1M Tris buffer, pH8.0) as substrate.

Microarray:

Article Title: Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
Article Snippet: Paragraph title: Replication timing profiling by microarray ... After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA.

Incubation:

Article Title: EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele
Article Snippet: .. For immunodetection, slides were blocked for 1 h in blocking solution (PBS, 1% BSA, 0.1% Tween20) and incubated overnight at 4°C with a mix of rat anti-BrdU monoclonal antibody (1:500 in blocking solution, AbD Serotec) that recognizes CldU, and mouse anti-BrdU monoclonal antibody (1:500 in blocking solution, Becton Dickinson) that recognizes IdU. .. Slides were washed three times with PBS and fixed with 4% formaldehyde for 10 min, following by three times washing with PBS and three times with blocking solution.

Article Title: Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells
Article Snippet: Cells were incubated with anti-RAD51 (Santa Cruz Biotechnology) for 1 h at room temperature in 1 × PBS/1% BSA/0.5% Tween. .. After washing, anti-rabbit phycoerythrin 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 7-amino-actinomycin D (7AAD) fluorescent dye (BD Biosciences).

Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior
Article Snippet: .. For fluorescent double labeling, that were performed to determine the cell phenotype, sections were incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1:200; Becton Dickinson), and a goat anti-DCX (1:200, Santa Cruz Laboratories). .. Antibodies were revealed with anti-mouse Alexa 488 and anti-goat Alexa 594 coupled antibodies (1:400; Molecular Probes).

Article Title: SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis
Article Snippet: Cells were incubated with α-H4K16Ac for 1 h at room temperature in 1× PBS/1% BSA/0.5% Tween. .. 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).

Article Title: PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA
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. .. After washing with PBS and 0.05% Tween 20 for 5 min three times, they were incubated with Alexa Fluor 555–conjugated goat anti–mouse IgG (1:500; Invitrogen) and Alexa Fluor 488–conjugated rabbit anti–rat IgG (1:500; Invitrogen) for 30 min at 37°C.

Article Title: Xenopus Cdc6 Performs Separate Functions in Initiating DNA Replication
Article Snippet: .. Briefly, cells were washed three times with PBS, fixed for 20 min (3.5% formaldehyde in PBS), permeabilized with 0.2% Triton X-100 for 20 min, and incubated with 10% fetal bovine serum in PBS for 1 h. GST was detected by staining with a rabbit polyclonal anti-GST antibody (Santa Cruz Biotechnology) at a dilution of 1:200 in PBS supplemented with 10% fetal bovine serum (F-PBS) for 2 h, followed by staining with AlexaFluor 488 goat anti-rabbit IgG conjugate (Molecular Probes, Eugene, OR) at a dilution of 1:1000 in F-PBS for 1 h. BrdU incorporated into DNA was detected by staining with a mouse monoclonal anti-BrdU antibody (BD PharMingen, San Diego, CA) at a dilution of 1:500 in F-PBS containing 125 U/ml benzon nuclease (Novagen, Madison, WI) for 2 h, followed by staining with AlexaFluor 594 goat anti-mouse IgG conjugate (Molecular Probes) at a dilution of 1:500 in F-PBS. .. The cells were then washed two times with PBS for 5 min each, and once with PBS containing 0.25 μg/ml Hoechst (Sigma Chemical) for 5 min. All incubations with antibody were done at room temperature.

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
Article Snippet: Primary antibodies were incubated for 1 h at 37°C followed by incubation with the appropriate combination of fluorescein isothiocyanate (FITC)-conjugated and rhodamine-conjugated anti-mouse and anti-rabbit secondary antibodies at 1:100 dilution for 30 min at 37°C for double-label assays. .. 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).

Article Title: Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design
Article Snippet: .. For each immunodetection, slides were first incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1∶200; Becton-Dickinson) or a goat anti-DCX (1∶400; Santa Cruz Laboratories). .. For chromogenic immunodetection, sections were then incubated for 1 h in biotin-conjugated species-specific secondary antibodies (1∶100; Vector Laboratories) followed by a peroxidase-avidin complex solution according to the manufacturer's protocol.

Activity Assay:

Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior
Article Snippet: The peroxidase activity of immune complexes was visualized with DAB staining using the VectaStain ABC kit (Vector Laboratories). .. For fluorescent double labeling, that were performed to determine the cell phenotype, sections were incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1:200; Becton Dickinson), and a goat anti-DCX (1:200, Santa Cruz Laboratories).

Article Title: Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex
Article Snippet: For BrdU immunostaining, sections were dewaxed in xylene, washed in ethanol and immersed in 3% hydrogen peroxide in methanol for 30 min at RT to block endogenous peroxidase activity. .. After blocking in 20% normal rabbit serum in TBS for 30 min, sections were immunostained using a mouse monoclonal anti BrdU antibody (Becton Dickinson, 347580; 1:200), followed by a biotinylated rabbit anti-mouse second antibody (Dako, E0354; 1:200) and then avidin-biotin horseradish peroxidase (HRP) (Dako, K0355; 1:1000) using the 3,3′-Diaminobenzidine (DAB) isopac system as substrate (Sigma, D9015).

Article Title: Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design
Article Snippet: For each immunodetection, slides were first incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1∶200; Becton-Dickinson) or a goat anti-DCX (1∶400; Santa Cruz Laboratories). .. The peroxidase activity of immune complexes was visualized with DAB staining using the VectaStain ABC kit (Vector Laboratories).

Expressing:

Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior
Article Snippet: Expression patterns of Alk2, Bmpr1a, Bmpr1b, Ctgf, Cxcr4, Ephb1, Etv1, Fezf2, Fz10, Id3, Msx2, Prox1, Rorb, Pou3f1/SCIP, Sorl1, Tbr1, Ttr1 , and Wnt3a genes were visualized by non-radioactive in situ hybridization ( ) to assess development of the cortical hem, dentate gyrus, other regions of the hippocampus, and overall brain structure. .. 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).

Modification:

Article Title: PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA
Article Snippet: Dynamic molecular combing and immunofluorescent detection Genomic DNA was prepared and combed onto the silanated coverslips (Matsunami Glass) as described previously ( ) with modification (see following paragraph). .. 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.

Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability
Article Snippet: The DNA fiber assay was performed as previously described ( , ), with slight modification. .. Spread DNA fibers were fixed and immunostained with anti-BrdU mouse monoclonal antibody (BD Biosciences) for IdU and anti-BrdU rat monoclonal antibody (Abcam) for CldU, detected with appropriate fluorescence-tagged secondary antibodies, and analyzed by IF microscopy.

Hybridization:

Article Title: Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
Article Snippet: After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA. .. Sample labeling, hybridization and data extraction were performed according to standard NimbleGen Systems procedures.

Flow Cytometry:

Article Title: Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells
Article Snippet: After washing, anti-rabbit phycoerythrin 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 7-amino-actinomycin D (7AAD) fluorescent dye (BD Biosciences). .. Data acquisition and analysis were performed using both a flow cytometer (cell counter FC500) and software (XL-MCL) from Beckman Coulter.

Article Title: SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis
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). .. Data acquisition and analysis were performed using both flow cytometer (cell counter FC500) and software (XL-MCL) from Beckman Coulter.

Article Title: Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
Article Snippet: These cells were resuspended in PBS containing 1% FBS, stained with propidium iodide (50 μg/ml) for 30 min in the presence of RNaseA (0.5 mg/ml), and then were sorted into early and late S phase fractions by flow cytometry. .. After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA.

Transferring:

Article Title: EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele
Article Snippet: 2 μl of cell suspension was dropped on the top of the microscope slide (Superfrost, Fisher Scientific), dryed for 6 min, 7 μl of spreading buffer (200 mM Tris–HCl pH 7.4, 50 mM EDTA, 0.5% SDS) was added to each drop, mixed by stirring with the pipette tip, followed by 2 min of incubation. .. For immunodetection, slides were blocked for 1 h in blocking solution (PBS, 1% BSA, 0.1% Tween20) and incubated overnight at 4°C with a mix of rat anti-BrdU monoclonal antibody (1:500 in blocking solution, AbD Serotec) that recognizes CldU, and mouse anti-BrdU monoclonal antibody (1:500 in blocking solution, Becton Dickinson) that recognizes IdU.

Injection:

Article Title: Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design
Article Snippet: Measurement of Hippocampal Neurogenesis Twenty-four hours after the injection of BrdU (120 mg per kg of body weight in a 300 µl bolus), mice were euthanized and transcardially perfused with 4% cold paraformaldehyde. .. For each immunodetection, slides were first incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1∶200; Becton-Dickinson) or a goat anti-DCX (1∶400; Santa Cruz Laboratories).

Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior
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). .. To label newborn neurons at P5, pups were injected with a single dose of BrdU (200 ug/g body weight) 24 h before euthanasia.

Light Microscopy:

Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior
Article Snippet: For fluorescent double labeling, that were performed to determine the cell phenotype, sections were incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1:200; Becton Dickinson), and a goat anti-DCX (1:200, Santa Cruz Laboratories). .. All BrdU-labeled cells in the granular cell layer and subgranular zone (SGZ) were counted in each section (n = 10 and 5 mice per group) at 400× magnification under a light microscope (Olympus) by an experimenter blinded to the study code.

Generated:

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
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). .. Rabbit anti-ORF K8 peptide PAb was generated by immunization with the N-terminal peptide (16-DNSEKDEAVIEED-28) bounded by N-Y and C-CS residues.

Affinity Purification:

Article Title: Replication stress induced site-specific phosphorylation targets WRN to the ubiquitin-proteasome pathway
Article Snippet: The phospho-specific antibodies were affinity-purified through a phospho-peptide-conjugated Sepharose CL-4B column. .. Commercially available anti-γH2AX mouse monoclonal antibody (Upstate Biotechnology), anti-RPA2 mouse monoclonal antibody (Calbiochem), anti-Rad51 rabbit polyclonal antibody (Calbiochem), anti-BrdU mouse monoclonal antibody (BD, 347580), and anti-Flag (M2) monoclonal (Sigma) antibodies were used.

Immunofluorescence:

Article Title: SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis
Article Snippet: Paragraph title: Immunofluorescence assays, BrdU treatment, FACs, and live cell experiments ... 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).

Article Title: Xenopus Cdc6 Performs Separate Functions in Initiating DNA Replication
Article Snippet: Paragraph title: Immunofluorescence ... Briefly, cells were washed three times with PBS, fixed for 20 min (3.5% formaldehyde in PBS), permeabilized with 0.2% Triton X-100 for 20 min, and incubated with 10% fetal bovine serum in PBS for 1 h. GST was detected by staining with a rabbit polyclonal anti-GST antibody (Santa Cruz Biotechnology) at a dilution of 1:200 in PBS supplemented with 10% fetal bovine serum (F-PBS) for 2 h, followed by staining with AlexaFluor 488 goat anti-rabbit IgG conjugate (Molecular Probes, Eugene, OR) at a dilution of 1:1000 in F-PBS for 1 h. BrdU incorporated into DNA was detected by staining with a mouse monoclonal anti-BrdU antibody (BD PharMingen, San Diego, CA) at a dilution of 1:500 in F-PBS containing 125 U/ml benzon nuclease (Novagen, Madison, WI) for 2 h, followed by staining with AlexaFluor 594 goat anti-mouse IgG conjugate (Molecular Probes) at a dilution of 1:500 in F-PBS.

Article Title: Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex
Article Snippet: Tissue processing and immunostaining Adrenal glands were fixed in either 4% paraformaldehyde (4 h) in PBS at 4°C for BrdU immunostaining, or 4% neutral buffered formalin (24 h) for dual-immunostaining and immunofluorescence. .. After blocking in 20% normal rabbit serum in TBS for 30 min, sections were immunostained using a mouse monoclonal anti BrdU antibody (Becton Dickinson, 347580; 1:200), followed by a biotinylated rabbit anti-mouse second antibody (Dako, E0354; 1:200) and then avidin-biotin horseradish peroxidase (HRP) (Dako, K0355; 1:1000) using the 3,3′-Diaminobenzidine (DAB) isopac system as substrate (Sigma, D9015).

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
Article Snippet: Paragraph title: IFA. ... 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).

Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior
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). .. To survey brain structure overall, histological assays included AChE histochemistry, a standard way to identify brain subdivisions ( ) and NeuN and β-tubulin immunohistochemistry (IHC), which allowed determination of neuron density and further identification of specific structures and cell groups.

Fluorescence:

Article Title: Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells
Article Snippet: For fluorescence activated cell sorting (FACS) analysis cells were washed and harvested after BrdU treatment and fixed on ice-cold 70% ethanol. .. After washing, anti-rabbit phycoerythrin 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 7-amino-actinomycin D (7AAD) fluorescent dye (BD Biosciences).

Article Title: Muscle regeneration is undisturbed by repeated polytraumatic injury
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. .. The BrdU positive cells selected by fluorescence were counted in a blind fashion with the software ImageJ (National Institute of Health, USA) in four fields of view at 63× magnification.

Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability
Article Snippet: .. Spread DNA fibers were fixed and immunostained with anti-BrdU mouse monoclonal antibody (BD Biosciences) for IdU and anti-BrdU rat monoclonal antibody (Abcam) for CldU, detected with appropriate fluorescence-tagged secondary antibodies, and analyzed by IF microscopy. .. To address the cell autonomous function of HINFP, we constructed a nonfunctional mutant mouse allele that expresses a C-terminally truncated, DNA binding-defective, and inactive transcription factor ( ).

Mutagenesis:

Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior
Article Snippet: Structural, gene expression, cell proliferation and cell death comparisons between the brains of double mutant mice and controls were based on at least 3 brains per group per age for each gene, protein product or histochemical assay. .. 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).

Isolation:

Article Title: Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
Article Snippet: After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA. .. Isolated early and late replicating DNA were amplified by whole-genome amplification (WGA) (Sigma-Aldrich, St Louis, MO, USA, GenomePlex), labeled with Cy3 and Cy5, and hybridized to a mouse whole-genome microarray (NimbleGen Symtems, Madison WIS, USA, 2006-07-26_MM8_WG_CGH or 100718_MM9_WG_CGH_HX3).

Immunohistochemistry:

Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior
Article Snippet: Every sixth sections, slices were processed for BrdU and DCX immunohistochemistry, as previously described ( ). .. For fluorescent double labeling, that were performed to determine the cell phenotype, sections were incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1:200; Becton Dickinson), and a goat anti-DCX (1:200, Santa Cruz Laboratories).

Article Title: Muscle regeneration is undisturbed by repeated polytraumatic injury
Article Snippet: Paragraph title: Immunohistochemistry ... 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.

Article Title: Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design
Article Snippet: Every sixth section throughout the hippocampus was processed for BrdU or DCX immunohistochemistry, as previously described . .. For each immunodetection, slides were first incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1∶200; Becton-Dickinson) or a goat anti-DCX (1∶400; Santa Cruz Laboratories).

Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior
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). .. To survey brain structure overall, histological assays included AChE histochemistry, a standard way to identify brain subdivisions ( ) and NeuN and β-tubulin immunohistochemistry (IHC), which allowed determination of neuron density and further identification of specific structures and cell groups.

Microscopy:

Article Title: EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele
Article Snippet: 2 μl of cell suspension was dropped on the top of the microscope slide (Superfrost, Fisher Scientific), dryed for 6 min, 7 μl of spreading buffer (200 mM Tris–HCl pH 7.4, 50 mM EDTA, 0.5% SDS) was added to each drop, mixed by stirring with the pipette tip, followed by 2 min of incubation. .. For immunodetection, slides were blocked for 1 h in blocking solution (PBS, 1% BSA, 0.1% Tween20) and incubated overnight at 4°C with a mix of rat anti-BrdU monoclonal antibody (1:500 in blocking solution, AbD Serotec) that recognizes CldU, and mouse anti-BrdU monoclonal antibody (1:500 in blocking solution, Becton Dickinson) that recognizes IdU.

Article Title: Muscle regeneration is undisturbed by repeated polytraumatic injury
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. .. To visualize the samples, a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany) was used.

Article Title: Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex
Article Snippet: Fixed adrenals were paraffin wax-embedded, cut and longitudinal 7-10 µm mid-sections mounted on polysine microscope slides. .. After blocking in 20% normal rabbit serum in TBS for 30 min, sections were immunostained using a mouse monoclonal anti BrdU antibody (Becton Dickinson, 347580; 1:200), followed by a biotinylated rabbit anti-mouse second antibody (Dako, E0354; 1:200) and then avidin-biotin horseradish peroxidase (HRP) (Dako, K0355; 1:1000) using the 3,3′-Diaminobenzidine (DAB) isopac system as substrate (Sigma, D9015).

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
Article Snippet: To visualize cellular chromatin, a drop (20 μl) of 4′, 6-diamidino-2-phenylindole (DAPI)-containing antifade slide mounting solution (Vector Shield) was added to the slide prior to microscopy. .. 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).

Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability
Article Snippet: .. Spread DNA fibers were fixed and immunostained with anti-BrdU mouse monoclonal antibody (BD Biosciences) for IdU and anti-BrdU rat monoclonal antibody (Abcam) for CldU, detected with appropriate fluorescence-tagged secondary antibodies, and analyzed by IF microscopy. .. To address the cell autonomous function of HINFP, we constructed a nonfunctional mutant mouse allele that expresses a C-terminally truncated, DNA binding-defective, and inactive transcription factor ( ).

Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior
Article Snippet: Images were captured with a Zeiss Axioskop microscope, Axiovision camera and software (Oberkochen, Germany). .. 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).

Immunodetection:

Article Title: EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele
Article Snippet: .. For immunodetection, slides were blocked for 1 h in blocking solution (PBS, 1% BSA, 0.1% Tween20) and incubated overnight at 4°C with a mix of rat anti-BrdU monoclonal antibody (1:500 in blocking solution, AbD Serotec) that recognizes CldU, and mouse anti-BrdU monoclonal antibody (1:500 in blocking solution, Becton Dickinson) that recognizes IdU. .. Slides were washed three times with PBS and fixed with 4% formaldehyde for 10 min, following by three times washing with PBS and three times with blocking solution.

Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior
Article Snippet: For chromogenic immunodetection, sections were then incubated for 1 h in biotin-conjugated species-specific secondary antibodies (1:100; Vector Laboratories) followed by a peroxidase-avidin complex solution according to the manufacturer’s protocol. .. For fluorescent double labeling, that were performed to determine the cell phenotype, sections were incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1:200; Becton Dickinson), and a goat anti-DCX (1:200, Santa Cruz Laboratories).

Article Title: PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA
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. .. After washing with PBS and 0.05% Tween 20 for 5 min three times, they were incubated with Alexa Fluor 555–conjugated goat anti–mouse IgG (1:500; Invitrogen) and Alexa Fluor 488–conjugated rabbit anti–rat IgG (1:500; Invitrogen) for 30 min at 37°C.

Article Title: Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design
Article Snippet: .. For each immunodetection, slides were first incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1∶200; Becton-Dickinson) or a goat anti-DCX (1∶400; Santa Cruz Laboratories). .. For chromogenic immunodetection, sections were then incubated for 1 h in biotin-conjugated species-specific secondary antibodies (1∶100; Vector Laboratories) followed by a peroxidase-avidin complex solution according to the manufacturer's protocol.

Immunostaining:

Article Title: Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex
Article Snippet: Paragraph title: Tissue processing and immunostaining ... After blocking in 20% normal rabbit serum in TBS for 30 min, sections were immunostained using a mouse monoclonal anti BrdU antibody (Becton Dickinson, 347580; 1:200), followed by a biotinylated rabbit anti-mouse second antibody (Dako, E0354; 1:200) and then avidin-biotin horseradish peroxidase (HRP) (Dako, K0355; 1:1000) using the 3,3′-Diaminobenzidine (DAB) isopac system as substrate (Sigma, D9015).

Staining:

Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior
Article Snippet: The peroxidase activity of immune complexes was visualized with DAB staining using the VectaStain ABC kit (Vector Laboratories). .. For fluorescent double labeling, that were performed to determine the cell phenotype, sections were incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1:200; Becton Dickinson), and a goat anti-DCX (1:200, Santa Cruz Laboratories).

Article Title: Muscle regeneration is undisturbed by repeated polytraumatic injury
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. .. An Alexa Fluor 488 anti-mouse secondary antibody and Alexa Fluor 546 anti-rabbit antibody (Molecular Probes, #A11001, #A11010) were used to visualize the labeling at 1:200 dilution and Hoechst nuclear staining (Invitrogen, #33342) at 1:1,000 dilution was used to label the nuclei of the cells.

Article Title: Xenopus Cdc6 Performs Separate Functions in Initiating DNA Replication
Article Snippet: .. Briefly, cells were washed three times with PBS, fixed for 20 min (3.5% formaldehyde in PBS), permeabilized with 0.2% Triton X-100 for 20 min, and incubated with 10% fetal bovine serum in PBS for 1 h. GST was detected by staining with a rabbit polyclonal anti-GST antibody (Santa Cruz Biotechnology) at a dilution of 1:200 in PBS supplemented with 10% fetal bovine serum (F-PBS) for 2 h, followed by staining with AlexaFluor 488 goat anti-rabbit IgG conjugate (Molecular Probes, Eugene, OR) at a dilution of 1:1000 in F-PBS for 1 h. BrdU incorporated into DNA was detected by staining with a mouse monoclonal anti-BrdU antibody (BD PharMingen, San Diego, CA) at a dilution of 1:500 in F-PBS containing 125 U/ml benzon nuclease (Novagen, Madison, WI) for 2 h, followed by staining with AlexaFluor 594 goat anti-mouse IgG conjugate (Molecular Probes) at a dilution of 1:500 in F-PBS. .. The cells were then washed two times with PBS for 5 min each, and once with PBS containing 0.25 μg/ml Hoechst (Sigma Chemical) for 5 min. All incubations with antibody were done at room temperature.

Article Title: Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
Article Snippet: These cells were resuspended in PBS containing 1% FBS, stained with propidium iodide (50 μg/ml) for 30 min in the presence of RNaseA (0.5 mg/ml), and then were sorted into early and late S phase fractions by flow cytometry. .. After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA.

Article Title: Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex
Article Snippet: After blocking in 20% normal rabbit serum in TBS for 30 min, sections were immunostained using a mouse monoclonal anti BrdU antibody (Becton Dickinson, 347580; 1:200), followed by a biotinylated rabbit anti-mouse second antibody (Dako, E0354; 1:200) and then avidin-biotin horseradish peroxidase (HRP) (Dako, K0355; 1:1000) using the 3,3′-Diaminobenzidine (DAB) isopac system as substrate (Sigma, D9015). .. After further HIER, Ki67 was detected using a monoclonal mouse antibody (Novocastra, NCL-Ki67-MM1; 1:250), followed by a biotinylated rabbit anti-mouse secondary antibody (Dako, E 0464; 1:500) and staining with streptavidin-HRP-DAB.

Article Title: Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design
Article Snippet: For each immunodetection, slides were first incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1∶200; Becton-Dickinson) or a goat anti-DCX (1∶400; Santa Cruz Laboratories). .. The peroxidase activity of immune complexes was visualized with DAB staining using the VectaStain ABC kit (Vector Laboratories).

Mouse Assay:

Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior
Article Snippet: For fluorescent double labeling, that were performed to determine the cell phenotype, sections were incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1:200; Becton Dickinson), and a goat anti-DCX (1:200, Santa Cruz Laboratories). .. All BrdU-labeled cells in the granular cell layer and subgranular zone (SGZ) were counted in each section (n = 10 and 5 mice per group) at 400× magnification under a light microscope (Olympus) by an experimenter blinded to the study code.

Article Title: Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design
Article Snippet: Measurement of Hippocampal Neurogenesis Twenty-four hours after the injection of BrdU (120 mg per kg of body weight in a 300 µl bolus), mice were euthanized and transcardially perfused with 4% cold paraformaldehyde. .. For each immunodetection, slides were first incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1∶200; Becton-Dickinson) or a goat anti-DCX (1∶400; Santa Cruz Laboratories).

Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior
Article Snippet: Structural, gene expression, cell proliferation and cell death comparisons between the brains of double mutant mice and controls were based on at least 3 brains per group per age for each gene, protein product or histochemical assay. .. 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).

In Situ Hybridization:

Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior
Article Snippet: Expression patterns of Alk2, Bmpr1a, Bmpr1b, Ctgf, Cxcr4, Ephb1, Etv1, Fezf2, Fz10, Id3, Msx2, Prox1, Rorb, Pou3f1/SCIP, Sorl1, Tbr1, Ttr1 , and Wnt3a genes were visualized by non-radioactive in situ hybridization ( ) to assess development of the cortical hem, dentate gyrus, other regions of the hippocampus, and overall brain structure. .. 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).

Plasmid Preparation:

Article Title: EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele
Article Snippet: For immunodetection, slides were blocked for 1 h in blocking solution (PBS, 1% BSA, 0.1% Tween20) and incubated overnight at 4°C with a mix of rat anti-BrdU monoclonal antibody (1:500 in blocking solution, AbD Serotec) that recognizes CldU, and mouse anti-BrdU monoclonal antibody (1:500 in blocking solution, Becton Dickinson) that recognizes IdU. .. For immunodetection, slides were blocked for 1 h in blocking solution (PBS, 1% BSA, 0.1% Tween20) and incubated overnight at 4°C with a mix of rat anti-BrdU monoclonal antibody (1:500 in blocking solution, AbD Serotec) that recognizes CldU, and mouse anti-BrdU monoclonal antibody (1:500 in blocking solution, Becton Dickinson) that recognizes IdU.

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

Article Title: Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex
Article Snippet: After blocking in 20% normal rabbit serum in TBS for 30 min, sections were immunostained using a mouse monoclonal anti BrdU antibody (Becton Dickinson, 347580; 1:200), followed by a biotinylated rabbit anti-mouse second antibody (Dako, E0354; 1:200) and then avidin-biotin horseradish peroxidase (HRP) (Dako, K0355; 1:1000) using the 3,3′-Diaminobenzidine (DAB) isopac system as substrate (Sigma, D9015). .. BrdU/Ki67 dual-immunostaining was carried out after dewaxing, blocking and HIER as described above, using a sheep polyclonal anti-BrdU antibody (Fitzgerald Industries, 20-BS17; 1:7000), followed by a biotinylated rabbit anti-sheep second antibody (Vector Labs, BA-6000; 1:500) and streptavidin-alkaline phosphatase (Dako, D0396; 1:1000) using Fast Blue (Sigma, F3378; 1mg/ml in 0.1M Tris buffer, pH8.0) as substrate.

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
Article Snippet: To visualize cellular chromatin, a drop (20 μl) of 4′, 6-diamidino-2-phenylindole (DAPI)-containing antifade slide mounting solution (Vector Shield) was added to the slide prior to microscopy. .. 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).

Article Title: Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design
Article Snippet: For each immunodetection, slides were first incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1∶200; Becton-Dickinson) or a goat anti-DCX (1∶400; Santa Cruz Laboratories). .. For each immunodetection, slides were first incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1∶200; Becton-Dickinson) or a goat anti-DCX (1∶400; Santa Cruz Laboratories).

Software:

Article Title: Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells
Article Snippet: After washing, anti-rabbit phycoerythrin 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 7-amino-actinomycin D (7AAD) fluorescent dye (BD Biosciences). .. Data acquisition and analysis were performed using both a flow cytometer (cell counter FC500) and software (XL-MCL) from Beckman Coulter.

Article Title: SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis
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). .. Data acquisition and analysis were performed using both flow cytometer (cell counter FC500) and software (XL-MCL) from Beckman Coulter.

Article Title: Muscle regeneration is undisturbed by repeated polytraumatic injury
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. .. The BrdU positive cells selected by fluorescence were counted in a blind fashion with the software ImageJ (National Institute of Health, USA) in four fields of view at 63× magnification.

Article Title: Xenopus Cdc6 Performs Separate Functions in Initiating DNA Replication
Article Snippet: Briefly, cells were washed three times with PBS, fixed for 20 min (3.5% formaldehyde in PBS), permeabilized with 0.2% Triton X-100 for 20 min, and incubated with 10% fetal bovine serum in PBS for 1 h. GST was detected by staining with a rabbit polyclonal anti-GST antibody (Santa Cruz Biotechnology) at a dilution of 1:200 in PBS supplemented with 10% fetal bovine serum (F-PBS) for 2 h, followed by staining with AlexaFluor 488 goat anti-rabbit IgG conjugate (Molecular Probes, Eugene, OR) at a dilution of 1:1000 in F-PBS for 1 h. BrdU incorporated into DNA was detected by staining with a mouse monoclonal anti-BrdU antibody (BD PharMingen, San Diego, CA) at a dilution of 1:500 in F-PBS containing 125 U/ml benzon nuclease (Novagen, Madison, WI) for 2 h, followed by staining with AlexaFluor 594 goat anti-mouse IgG conjugate (Molecular Probes) at a dilution of 1:500 in F-PBS. .. All images were captured at identical magnification and exposure times using a Quantix cooled charge-coupled device camera (Photometrics, Tucson, AZ) with Isee software (Inovision, Raleigh, NC).

Article Title: The Mcm2–7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing
Article Snippet: After DNA was denatured with formamide, IdU-labeled DNA was detected with mouse anti-BrdU monoclonal antibody (BD Biosciences) and Alexa Fluor 555-conjugated goat anti-mouse IgG (Thermo Fisher). .. Fluorescent signals were measured by using MetaMorph version 6.1 software (MDS Analytical Technologies) and converted to kilobase pairs using a conversion factor of 1 μm = 2.32 ± 0.11 kbp ( ).

Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior
Article Snippet: Images were captured with a Zeiss Axioskop microscope, Axiovision camera and software (Oberkochen, Germany). .. 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).

Avidin-Biotin Assay:

Article Title: Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex
Article Snippet: .. After blocking in 20% normal rabbit serum in TBS for 30 min, sections were immunostained using a mouse monoclonal anti BrdU antibody (Becton Dickinson, 347580; 1:200), followed by a biotinylated rabbit anti-mouse second antibody (Dako, E0354; 1:200) and then avidin-biotin horseradish peroxidase (HRP) (Dako, K0355; 1:1000) using the 3,3′-Diaminobenzidine (DAB) isopac system as substrate (Sigma, D9015). .. BrdU/Ki67 dual-immunostaining was carried out after dewaxing, blocking and HIER as described above, using a sheep polyclonal anti-BrdU antibody (Fitzgerald Industries, 20-BS17; 1:7000), followed by a biotinylated rabbit anti-sheep second antibody (Vector Labs, BA-6000; 1:500) and streptavidin-alkaline phosphatase (Dako, D0396; 1:1000) using Fast Blue (Sigma, F3378; 1mg/ml in 0.1M Tris buffer, pH8.0) as substrate.

Labeling:

Article Title: EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele
Article Snippet: For replication restart, cells were treated with 1 mM hydroxyurea (HU, Sigma–Aldrich) for 15 min after 20 min CldU labelling, followed by 40 min IdU labeling. .. For immunodetection, slides were blocked for 1 h in blocking solution (PBS, 1% BSA, 0.1% Tween20) and incubated overnight at 4°C with a mix of rat anti-BrdU monoclonal antibody (1:500 in blocking solution, AbD Serotec) that recognizes CldU, and mouse anti-BrdU monoclonal antibody (1:500 in blocking solution, Becton Dickinson) that recognizes IdU.

Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior
Article Snippet: .. For fluorescent double labeling, that were performed to determine the cell phenotype, sections were incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1:200; Becton Dickinson), and a goat anti-DCX (1:200, Santa Cruz Laboratories). .. Antibodies were revealed with anti-mouse Alexa 488 and anti-goat Alexa 594 coupled antibodies (1:400; Molecular Probes).

Article Title: PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA
Article Snippet: NU1025 was pretreated for 2 h before IdU labeling. .. 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.

Article Title: Muscle regeneration is undisturbed by repeated polytraumatic injury
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. .. An Alexa Fluor 488 anti-mouse secondary antibody and Alexa Fluor 546 anti-rabbit antibody (Molecular Probes, #A11001, #A11010) were used to visualize the labeling at 1:200 dilution and Hoechst nuclear staining (Invitrogen, #33342) at 1:1,000 dilution was used to label the nuclei of the cells.

Article Title: Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
Article Snippet: In brief, cells were labeled with 50 μM BrdU for 2 h, washed twice with ice-cold PBS, trypsinized, and then were fixed in 75% ethanol. .. After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA.

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
Article Snippet: Rhodamine-conjugated anti-mouse secondary antibody was diluted at 1:100 for single labeling. .. 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).

Article Title: The Mcm2–7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing
Article Snippet: Cells were pulse-labeled with 100 μ m IdU for 20 min, washed twice with PBS, and then labeled with 100 μ m CldU for 20 min. Genomic DNA was prepared and combed onto the silanated coverslips (Matsunami Glass) as described previously ( ). .. After DNA was denatured with formamide, IdU-labeled DNA was detected with mouse anti-BrdU monoclonal antibody (BD Biosciences) and Alexa Fluor 555-conjugated goat anti-mouse IgG (Thermo Fisher).

Article Title: BMP signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior
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). .. Caspase-3 IHC was used to identify apoptotic cells; and phospho Histone H3 (PH3) IHC and 5-bromo-2-deoxyuridine (BrdU) labeling to identify cells in division.

Preserving:

Article Title: SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis
Article Snippet: Chromatin BrdU incorporation was detected by incubation for 45 min at 37°C in antibody/nuclease solution (Amersham Biosciences) that improves preservation of antigens. .. 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).

BrdU Incorporation Assay:

Article Title: SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis
Article Snippet: Chromatin BrdU incorporation was detected by incubation for 45 min at 37°C in antibody/nuclease solution (Amersham Biosciences) that improves preservation of antigens. .. 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).

Immunoprecipitation:

Article Title: Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains
Article Snippet: .. After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA. .. Isolated early and late replicating DNA were amplified by whole-genome amplification (WGA) (Sigma-Aldrich, St Louis, MO, USA, GenomePlex), labeled with Cy3 and Cy5, and hybridized to a mouse whole-genome microarray (NimbleGen Symtems, Madison WIS, USA, 2006-07-26_MM8_WG_CGH or 100718_MM9_WG_CGH_HX3).

Marker:

Article Title: Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design
Article Snippet: For each immunodetection, slides were first incubated overnight at 4°C with a mouse monoclonal anti-BrdU antibody (1∶200; Becton-Dickinson) or a goat anti-DCX (1∶400; Santa Cruz Laboratories). .. For fluorescent double-labeling, performed to determine the cell phenotype, sections were incubated overnight at 4°C with anti-sheep BrdU (1∶200, Interchim), anti-goat DCX (1∶200, Santa Cruz Laboratories), or an anti-GFAP (glial fibrillary acidic protein, marker for astrocytes, 1∶250, Dako).

FACS:

Article Title: Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells
Article Snippet: For fluorescence activated cell sorting (FACS) analysis cells were washed and harvested after BrdU treatment and fixed on ice-cold 70% ethanol. .. After washing, anti-rabbit phycoerythrin 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 7-amino-actinomycin D (7AAD) fluorescent dye (BD Biosciences).

Article Title: SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis
Article Snippet: Paragraph title: Immunofluorescence assays, BrdU treatment, FACs, and live cell experiments ... 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).

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  • 96
    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
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    Becton Dickinson anti idu
    Cross-reactivity and validation of antibody staining. Section of an embryo exposed to <t>IdU</t> shows specific staining using an antibody against IdU (Panel A). When an antibody against <t>CldU</t> is used there is no aspecific staining visible (Panel C). When an embryo is exposed to CldU and an antibody agains IdU is used some cross reactivity is observed (Panel B). Panel D shows specific staining for CldU. Abbreviations: ift: Inflow Tract; nt: Neural Tubel; oft: Outflow Tract; v: Ventricle. Panel E shows the relation between the number of nuclei labelled for IdU and for CldU at equal exposure times. Each point represents a section. There was no significant difference between 2 and 4 hours of exposure time. The linear relation shows a high correlation coefficient (R 2 = 0.991) and detection of 7.2% less IdU than CldU positive nuclei.
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    Becton Dickinson anti brdu r pe conjugated mouse mab
    Rate of cellular division of EGFP-positive and EGFP-negative cells in HeLa cell culture infected with the BoHV-4 V.test EGFP Xho I strain. HeLa cells were mock infected (A and B) or infected with the BoHV-4 V.test EGFP Xho I strain at an MOI of 0.5 PFU/cell (C and D). The cells were then passaged every other day for 8 days (1:2 split ratio). At 9 days postinfection, the cells were mock pulsed (A and C) or pulsed with <t>BrdU</t> (B and D) for 1 h as described in Materials and Methods. Cells positive for the incorporation of BrdU were then revealed by immunofluorescence staining with <t>anti-BrdU-R-PE</t> and analyzed by flow cytometry for the emission of green (EGFP) and red (anti-BrdU-R-PE) signals. By using the sample of infected cells pulsed with BrdU (D), the rate of BrdU-positive cells was estimated for 10,000 EGFP-negative (E) or EGFP-positive (F) cells.
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    Becton Dickinson anti cd1d
    <t>CD1d</t> trafficking through early endosomes, late endosomes, and lysosomes. (A–C) In double-positive (DP) thymocytes, super-resolution microscopy was used to determine intracellular localization of CD1d molecules in early endosomes, late endosomes, and lysosomes visualized in the red channel by early endosome antigen 1 (EEA1), Rab7, and lysosome-associated membrane protein 1 (LAMP1), respectively. Co-localization areas were presented in white (right panels). DAPI was applied to visualize the nucleus (bar = 5 µm). (D) DP thymocytes were analyzed for co-localization between green and red signals using the ImageJ’s co-localization plugin, and the ratio of co-localized and total green area was plotted and statistically analyzed using the unpaired t-test. Although a significant shift from late to toward early endosomes could be observed in Vav Cre GCS f/f DP thymocytes, the amount of CD1d in lysosomes was equal. Shown are means ± SEM, N = 20 cells per group. (E) A tendency toward less but larger LAMP1 + lysosomes could be seen in DP thymocytes of Vav Cre GCS f/f mice, however, the difference was not statistically significant. The bars show means ± SEM, N = 7 cells per group.
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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

    Cross-reactivity and validation of antibody staining. Section of an embryo exposed to IdU shows specific staining using an antibody against IdU (Panel A). When an antibody against CldU is used there is no aspecific staining visible (Panel C). When an embryo is exposed to CldU and an antibody agains IdU is used some cross reactivity is observed (Panel B). Panel D shows specific staining for CldU. Abbreviations: ift: Inflow Tract; nt: Neural Tubel; oft: Outflow Tract; v: Ventricle. Panel E shows the relation between the number of nuclei labelled for IdU and for CldU at equal exposure times. Each point represents a section. There was no significant difference between 2 and 4 hours of exposure time. The linear relation shows a high correlation coefficient (R 2 = 0.991) and detection of 7.2% less IdU than CldU positive nuclei.

    Journal: PLoS ONE

    Article Title: Measurement and 3D-Visualization of Cell-Cycle Length Using Double Labelling with Two Thymidine Analogues Applied in Early Heart Development

    doi: 10.1371/journal.pone.0047719

    Figure Lengend Snippet: Cross-reactivity and validation of antibody staining. Section of an embryo exposed to IdU shows specific staining using an antibody against IdU (Panel A). When an antibody against CldU is used there is no aspecific staining visible (Panel C). When an embryo is exposed to CldU and an antibody agains IdU is used some cross reactivity is observed (Panel B). Panel D shows specific staining for CldU. Abbreviations: ift: Inflow Tract; nt: Neural Tubel; oft: Outflow Tract; v: Ventricle. Panel E shows the relation between the number of nuclei labelled for IdU and for CldU at equal exposure times. Each point represents a section. There was no significant difference between 2 and 4 hours of exposure time. The linear relation shows a high correlation coefficient (R 2 = 0.991) and detection of 7.2% less IdU than CldU positive nuclei.

    Article Snippet: Each section was exposed overnight to a mixture of anti-IdU (mouse-monoclonal anti-BrdU; BD, 347580), anti-CldU (rat-monoclonal anti-BrdU; Serotec, OBT0030CX) and anti-cTnI (rabbit polyclonal; HyTest, 4T21/2) followed by incubation for at least 2 hrs with a mixture of the fluorescent antibodies, goat-anti-mouse-Alexa 680, goat-anti-rat-Alexa 568, goat-anti-rabbit-Alexa 405 (Invitrogen), and Sytox green 488 (Invitrogen).

    Techniques: Staining

    Image analysis and visualisation. Panel A. Using a Sytox green staining, all nuclei are first detected based on a local-maxima threshold. All detected objects that were at least twice as large as the median object size were processed to separate these fused nuclei (inserts). Panel B shows a schematic overview of the image processing steps involved in the recognition of IdU- and CldU-positive nuclei. After the detection of the Sytox green stained nuclei, each nucleus is individually processed. A zone is selected around all nuclei which will be excluded in the following measurement (gray zone). For each nucleus within the region of interest (myocardium), the algorithm measures the signal in (red area) and around (green area) the nucleus in the IdU and CldU channels. The measurement of the local background excludes the locations at which other nuclei were detected (gray zone). When the signal in the nucleus is at least a standard deviation above the background, the nucleus is classified as positively labelled. The program generates control images both for the nuclei detection as well as for which nuclei are positive for the proliferation markers. The difference between the two proliferation markers is used to determine ΔF (number of green nuclei divided by total number of nuclei). Panel C shows how the quantitative information can be projected onto a reconstruction or onto the original section. Each unit in the boxel representation has a volume of approximately 21 3 µm 3 , and is the central boxel of the sampling volume of approximately 105 3 µm 3 that is required for reliable measurement of the labelling indices [15] .

    Journal: PLoS ONE

    Article Title: Measurement and 3D-Visualization of Cell-Cycle Length Using Double Labelling with Two Thymidine Analogues Applied in Early Heart Development

    doi: 10.1371/journal.pone.0047719

    Figure Lengend Snippet: Image analysis and visualisation. Panel A. Using a Sytox green staining, all nuclei are first detected based on a local-maxima threshold. All detected objects that were at least twice as large as the median object size were processed to separate these fused nuclei (inserts). Panel B shows a schematic overview of the image processing steps involved in the recognition of IdU- and CldU-positive nuclei. After the detection of the Sytox green stained nuclei, each nucleus is individually processed. A zone is selected around all nuclei which will be excluded in the following measurement (gray zone). For each nucleus within the region of interest (myocardium), the algorithm measures the signal in (red area) and around (green area) the nucleus in the IdU and CldU channels. The measurement of the local background excludes the locations at which other nuclei were detected (gray zone). When the signal in the nucleus is at least a standard deviation above the background, the nucleus is classified as positively labelled. The program generates control images both for the nuclei detection as well as for which nuclei are positive for the proliferation markers. The difference between the two proliferation markers is used to determine ΔF (number of green nuclei divided by total number of nuclei). Panel C shows how the quantitative information can be projected onto a reconstruction or onto the original section. Each unit in the boxel representation has a volume of approximately 21 3 µm 3 , and is the central boxel of the sampling volume of approximately 105 3 µm 3 that is required for reliable measurement of the labelling indices [15] .

    Article Snippet: Each section was exposed overnight to a mixture of anti-IdU (mouse-monoclonal anti-BrdU; BD, 347580), anti-CldU (rat-monoclonal anti-BrdU; Serotec, OBT0030CX) and anti-cTnI (rabbit polyclonal; HyTest, 4T21/2) followed by incubation for at least 2 hrs with a mixture of the fluorescent antibodies, goat-anti-mouse-Alexa 680, goat-anti-rat-Alexa 568, goat-anti-rabbit-Alexa 405 (Invitrogen), and Sytox green 488 (Invitrogen).

    Techniques: Staining, Standard Deviation, Sampling

    Application in heart development. 3D visualisation of cell cycle length in the heart at stages HH9 (Panel A), HH12 (Panel B) and HH16 (Panel C) of chicken embryonic development. The pointer in panel B indicates the region with a high proliferation rate at the site of early ventricle formation. Panel C shows the quantitative reconstructions of the individual labelling indices for CldU and IdU, on which the cycle lengths are based. The pointers in the CldU reconstruction indicate areas in which a low fraction of cells is positive in both CldU and IdU reconstructions, resulting in a low labelling difference and thus a long cell cycle length. The pointers in the IdU reconstruction show large differences in IdU and CldU labelling indices, indicating short cell cycle lengths. Note the heterogeneity in cell cycle lengths in different parts of the heart at every stage. Interactive versions of the 3D-reconstructions can be found in Interactive 3D-pdf S1 .

    Journal: PLoS ONE

    Article Title: Measurement and 3D-Visualization of Cell-Cycle Length Using Double Labelling with Two Thymidine Analogues Applied in Early Heart Development

    doi: 10.1371/journal.pone.0047719

    Figure Lengend Snippet: Application in heart development. 3D visualisation of cell cycle length in the heart at stages HH9 (Panel A), HH12 (Panel B) and HH16 (Panel C) of chicken embryonic development. The pointer in panel B indicates the region with a high proliferation rate at the site of early ventricle formation. Panel C shows the quantitative reconstructions of the individual labelling indices for CldU and IdU, on which the cycle lengths are based. The pointers in the CldU reconstruction indicate areas in which a low fraction of cells is positive in both CldU and IdU reconstructions, resulting in a low labelling difference and thus a long cell cycle length. The pointers in the IdU reconstruction show large differences in IdU and CldU labelling indices, indicating short cell cycle lengths. Note the heterogeneity in cell cycle lengths in different parts of the heart at every stage. Interactive versions of the 3D-reconstructions can be found in Interactive 3D-pdf S1 .

    Article Snippet: Each section was exposed overnight to a mixture of anti-IdU (mouse-monoclonal anti-BrdU; BD, 347580), anti-CldU (rat-monoclonal anti-BrdU; Serotec, OBT0030CX) and anti-cTnI (rabbit polyclonal; HyTest, 4T21/2) followed by incubation for at least 2 hrs with a mixture of the fluorescent antibodies, goat-anti-mouse-Alexa 680, goat-anti-rat-Alexa 568, goat-anti-rabbit-Alexa 405 (Invitrogen), and Sytox green 488 (Invitrogen).

    Techniques:

    Rate of cellular division of EGFP-positive and EGFP-negative cells in HeLa cell culture infected with the BoHV-4 V.test EGFP Xho I strain. HeLa cells were mock infected (A and B) or infected with the BoHV-4 V.test EGFP Xho I strain at an MOI of 0.5 PFU/cell (C and D). The cells were then passaged every other day for 8 days (1:2 split ratio). At 9 days postinfection, the cells were mock pulsed (A and C) or pulsed with BrdU (B and D) for 1 h as described in Materials and Methods. Cells positive for the incorporation of BrdU were then revealed by immunofluorescence staining with anti-BrdU-R-PE and analyzed by flow cytometry for the emission of green (EGFP) and red (anti-BrdU-R-PE) signals. By using the sample of infected cells pulsed with BrdU (D), the rate of BrdU-positive cells was estimated for 10,000 EGFP-negative (E) or EGFP-positive (F) cells.

    Journal: Journal of Virology

    Article Title: Investigation of the Susceptibility of Human Cell Lines to Bovine Herpesvirus 4 Infection: Demonstration that Human Cells Can Support a Nonpermissive Persistent Infection Which Protects Them against Tumor Necrosis Factor Alpha-Induced Apoptosis

    doi: 10.1128/JVI.78.5.2336-2347.2004

    Figure Lengend Snippet: Rate of cellular division of EGFP-positive and EGFP-negative cells in HeLa cell culture infected with the BoHV-4 V.test EGFP Xho I strain. HeLa cells were mock infected (A and B) or infected with the BoHV-4 V.test EGFP Xho I strain at an MOI of 0.5 PFU/cell (C and D). The cells were then passaged every other day for 8 days (1:2 split ratio). At 9 days postinfection, the cells were mock pulsed (A and C) or pulsed with BrdU (B and D) for 1 h as described in Materials and Methods. Cells positive for the incorporation of BrdU were then revealed by immunofluorescence staining with anti-BrdU-R-PE and analyzed by flow cytometry for the emission of green (EGFP) and red (anti-BrdU-R-PE) signals. By using the sample of infected cells pulsed with BrdU (D), the rate of BrdU-positive cells was estimated for 10,000 EGFP-negative (E) or EGFP-positive (F) cells.

    Article Snippet: BrdU staining was then performed by adding of 50 μl of anti-BrdU-R-PE-conjugated mouse MAb (Becton Dickinson) for 45 min at room temperature.

    Techniques: Cell Culture, Infection, Immunofluorescence, Staining, Flow Cytometry, Cytometry

    CD1d trafficking through early endosomes, late endosomes, and lysosomes. (A–C) In double-positive (DP) thymocytes, super-resolution microscopy was used to determine intracellular localization of CD1d molecules in early endosomes, late endosomes, and lysosomes visualized in the red channel by early endosome antigen 1 (EEA1), Rab7, and lysosome-associated membrane protein 1 (LAMP1), respectively. Co-localization areas were presented in white (right panels). DAPI was applied to visualize the nucleus (bar = 5 µm). (D) DP thymocytes were analyzed for co-localization between green and red signals using the ImageJ’s co-localization plugin, and the ratio of co-localized and total green area was plotted and statistically analyzed using the unpaired t-test. Although a significant shift from late to toward early endosomes could be observed in Vav Cre GCS f/f DP thymocytes, the amount of CD1d in lysosomes was equal. Shown are means ± SEM, N = 20 cells per group. (E) A tendency toward less but larger LAMP1 + lysosomes could be seen in DP thymocytes of Vav Cre GCS f/f mice, however, the difference was not statistically significant. The bars show means ± SEM, N = 7 cells per group.

    Journal: Frontiers in Immunology

    Article Title: Glucosylceramide Synthase Is Involved in Development of Invariant Natural Killer T Cells

    doi: 10.3389/fimmu.2017.00848

    Figure Lengend Snippet: CD1d trafficking through early endosomes, late endosomes, and lysosomes. (A–C) In double-positive (DP) thymocytes, super-resolution microscopy was used to determine intracellular localization of CD1d molecules in early endosomes, late endosomes, and lysosomes visualized in the red channel by early endosome antigen 1 (EEA1), Rab7, and lysosome-associated membrane protein 1 (LAMP1), respectively. Co-localization areas were presented in white (right panels). DAPI was applied to visualize the nucleus (bar = 5 µm). (D) DP thymocytes were analyzed for co-localization between green and red signals using the ImageJ’s co-localization plugin, and the ratio of co-localized and total green area was plotted and statistically analyzed using the unpaired t-test. Although a significant shift from late to toward early endosomes could be observed in Vav Cre GCS f/f DP thymocytes, the amount of CD1d in lysosomes was equal. Shown are means ± SEM, N = 20 cells per group. (E) A tendency toward less but larger LAMP1 + lysosomes could be seen in DP thymocytes of Vav Cre GCS f/f mice, however, the difference was not statistically significant. The bars show means ± SEM, N = 7 cells per group.

    Article Snippet: The following monoclonal antibodies were used: anti-CD1d (clone: 1B1); anti-CD3ε (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD11c (HL3), anti-CD19 (MB19-1), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-CD150/SLAM (9D1), anti-Ly108 (13G3), anti-MHCII (M5/144.15.2), anti-NK1.1 (PK136), anti-TCR-Vβ2 (B20.6), anti-TCR-Vβ7 (TR310), and anti-TCR-Vβ8.1 and 8.2 (MR5-2) from BD, Heidelberg, Germany, Biolegend, San Diego, CA, USA, and eBioscience, San Diego, CA, USA.

    Techniques: Microscopy, Mouse Assay

    Antigen presentation and recognition in Vav Cre GCS f/f mice. (A,B) Double-positive (DP) thymocytes were tested for their antigen presentation capacity toward invariant natural killer T (iNKT) cells in vitro . To this end, iNKT-depleted wild-type (WT), Vav Cre GCS f/f , and CD1d –/– DP thymocytes were exposed to increasing concentrations of αGalCer and co-incubated with responder WT iNKT cells enriched from livers of TCRVα14-Jα281 transgenic mice. The activation measured as secretion of IFNγ (A) and IL4 (B) did not differ between WT and Vav Cre GCS f/f DP thymocytes. CD1d –/– DP thymocytes served as negative controls, and the corresponding bars cannot be discriminated from the zero line in all but one concentration. Shown are means ± SEM, N = 6–9 per group. (C) Activation of iNKT cells was tested in vivo . WT and Vav Cre GCS f/f mice were i.p. injected with either 0.2 or 3 µg αGalCer. Eight hours later, splenic iNKT cells were analyzed for surface CD69 expression by flow cytometry by gating on CD19 − /PBS57-CD1d + /CD44 + lymphocytes. Expression of CD69 did not differ between WT and Vav Cre GCS f/f DP thymocytes. In parallel, serum was analyzed for IFNγ and IL4 levels. In Vav Cre GCS f/f mice injected with 3 µg αGalCer, IFNγ levels were significantly lower than in the WT controls. All other measurements did not show a statistically significant difference. Shown are means ± SEM, N = 3 per group. (D) Activation of iNKT cells was tested in vitro . iNKT cells from livers and spleens of WT and Vav Cre GCS f/f mice were exposed to αGalCer-loaded WT DP thymocytes. The activation measured as IFNγ secretion did not differ between WT and Vav Cre GCS f/f iNKT cells. Shown are means ± SEM, n = 3–6 per group. (E) Splenic conventional T cells were tested for their T cell receptor (TCR)-independent and TCR-dependent activation in vitro . WT and Vav Cre GCS f/f splenic T cells were activated by PMA/calcium ionophore A23187 or by plate-bound anti-CD3/anti-CD28 antibodies. Vehicle (media)-treated cells served as controls. No statistically significant differences could be found in the IFNγ secretion between WT and Vav Cre GCS f/f T cells. Shown are means ± SEM, n = 6 per group.

    Journal: Frontiers in Immunology

    Article Title: Glucosylceramide Synthase Is Involved in Development of Invariant Natural Killer T Cells

    doi: 10.3389/fimmu.2017.00848

    Figure Lengend Snippet: Antigen presentation and recognition in Vav Cre GCS f/f mice. (A,B) Double-positive (DP) thymocytes were tested for their antigen presentation capacity toward invariant natural killer T (iNKT) cells in vitro . To this end, iNKT-depleted wild-type (WT), Vav Cre GCS f/f , and CD1d –/– DP thymocytes were exposed to increasing concentrations of αGalCer and co-incubated with responder WT iNKT cells enriched from livers of TCRVα14-Jα281 transgenic mice. The activation measured as secretion of IFNγ (A) and IL4 (B) did not differ between WT and Vav Cre GCS f/f DP thymocytes. CD1d –/– DP thymocytes served as negative controls, and the corresponding bars cannot be discriminated from the zero line in all but one concentration. Shown are means ± SEM, N = 6–9 per group. (C) Activation of iNKT cells was tested in vivo . WT and Vav Cre GCS f/f mice were i.p. injected with either 0.2 or 3 µg αGalCer. Eight hours later, splenic iNKT cells were analyzed for surface CD69 expression by flow cytometry by gating on CD19 − /PBS57-CD1d + /CD44 + lymphocytes. Expression of CD69 did not differ between WT and Vav Cre GCS f/f DP thymocytes. In parallel, serum was analyzed for IFNγ and IL4 levels. In Vav Cre GCS f/f mice injected with 3 µg αGalCer, IFNγ levels were significantly lower than in the WT controls. All other measurements did not show a statistically significant difference. Shown are means ± SEM, N = 3 per group. (D) Activation of iNKT cells was tested in vitro . iNKT cells from livers and spleens of WT and Vav Cre GCS f/f mice were exposed to αGalCer-loaded WT DP thymocytes. The activation measured as IFNγ secretion did not differ between WT and Vav Cre GCS f/f iNKT cells. Shown are means ± SEM, n = 3–6 per group. (E) Splenic conventional T cells were tested for their T cell receptor (TCR)-independent and TCR-dependent activation in vitro . WT and Vav Cre GCS f/f splenic T cells were activated by PMA/calcium ionophore A23187 or by plate-bound anti-CD3/anti-CD28 antibodies. Vehicle (media)-treated cells served as controls. No statistically significant differences could be found in the IFNγ secretion between WT and Vav Cre GCS f/f T cells. Shown are means ± SEM, n = 6 per group.

    Article Snippet: The following monoclonal antibodies were used: anti-CD1d (clone: 1B1); anti-CD3ε (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD11c (HL3), anti-CD19 (MB19-1), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-CD150/SLAM (9D1), anti-Ly108 (13G3), anti-MHCII (M5/144.15.2), anti-NK1.1 (PK136), anti-TCR-Vβ2 (B20.6), anti-TCR-Vβ7 (TR310), and anti-TCR-Vβ8.1 and 8.2 (MR5-2) from BD, Heidelberg, Germany, Biolegend, San Diego, CA, USA, and eBioscience, San Diego, CA, USA.

    Techniques: Mouse Assay, In Vitro, Incubation, Transgenic Assay, Activation Assay, Concentration Assay, In Vivo, Injection, Expressing, Flow Cytometry, Cytometry

    Vav Cre GCS f/f mice showed a typical thymocyte development and an unaltered expression of CD1d, SLAM and Ly108 molecules. (A) No statistically significant differences were observed between 8 week old WT and Vav Cre GCS f/f mice in terms of body weight and the weight and cellularity of spleen and thymus. Bars show means ± SEM, N = 6 per group. (B) Thymocyte development was investigated in 8 week old mice by flow cytometry using antibodies against CD4 and CD8. The double-negative (DN) stage was further subdivided into DN1 (CD25 – /CD44 + ), DN2 (CD25 + /CD44 + ), DN3 (CD25 + /CD44 – ), and DN4 (CD25 – /CD44 – ). Representative dot plots (upper panels) as well as relative and absolute numbers (lower panels) are shown (mean ± SEM, N = 13 per group). No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice. (C) Frequencies of B (CD3 – /CD19 + ) and T (CD3 + /CD19 – ) cells were measured by flow cytometry in spleens of 8 week old mice. Representative dot-plots and quantifications are shown (mean ± SEM, N = 13 per group). No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice. (D,E) Expression of CD1d was measured on DP (CD4 + /CD8 + ) thymocytes (D) and splenic DC (CD11c + /MHCII + ) (E) and expressed as mean fluorescence intensity (MFI). No statistically significant difference could be observed between WT and Vav Cre GCS f/f mice. Shown are means ± SEM, N = 6 per group in panel (D) and 4 per group in panel (E) . (F,G) Expression of SLAM (CD150) and Ly108, respectively, was measured on DP (CD4 + /CD8 + ) thymocytes and expressed as MFI. No statistically significant difference could be observed between WT and Vav Cre GCS f/f mice. Shown are means ± SEM, N = 4 per group.

    Journal: Frontiers in Immunology

    Article Title: Glucosylceramide Synthase Is Involved in Development of Invariant Natural Killer T Cells

    doi: 10.3389/fimmu.2017.00848

    Figure Lengend Snippet: Vav Cre GCS f/f mice showed a typical thymocyte development and an unaltered expression of CD1d, SLAM and Ly108 molecules. (A) No statistically significant differences were observed between 8 week old WT and Vav Cre GCS f/f mice in terms of body weight and the weight and cellularity of spleen and thymus. Bars show means ± SEM, N = 6 per group. (B) Thymocyte development was investigated in 8 week old mice by flow cytometry using antibodies against CD4 and CD8. The double-negative (DN) stage was further subdivided into DN1 (CD25 – /CD44 + ), DN2 (CD25 + /CD44 + ), DN3 (CD25 + /CD44 – ), and DN4 (CD25 – /CD44 – ). Representative dot plots (upper panels) as well as relative and absolute numbers (lower panels) are shown (mean ± SEM, N = 13 per group). No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice. (C) Frequencies of B (CD3 – /CD19 + ) and T (CD3 + /CD19 – ) cells were measured by flow cytometry in spleens of 8 week old mice. Representative dot-plots and quantifications are shown (mean ± SEM, N = 13 per group). No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice. (D,E) Expression of CD1d was measured on DP (CD4 + /CD8 + ) thymocytes (D) and splenic DC (CD11c + /MHCII + ) (E) and expressed as mean fluorescence intensity (MFI). No statistically significant difference could be observed between WT and Vav Cre GCS f/f mice. Shown are means ± SEM, N = 6 per group in panel (D) and 4 per group in panel (E) . (F,G) Expression of SLAM (CD150) and Ly108, respectively, was measured on DP (CD4 + /CD8 + ) thymocytes and expressed as MFI. No statistically significant difference could be observed between WT and Vav Cre GCS f/f mice. Shown are means ± SEM, N = 4 per group.

    Article Snippet: The following monoclonal antibodies were used: anti-CD1d (clone: 1B1); anti-CD3ε (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD11c (HL3), anti-CD19 (MB19-1), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-CD150/SLAM (9D1), anti-Ly108 (13G3), anti-MHCII (M5/144.15.2), anti-NK1.1 (PK136), anti-TCR-Vβ2 (B20.6), anti-TCR-Vβ7 (TR310), and anti-TCR-Vβ8.1 and 8.2 (MR5-2) from BD, Heidelberg, Germany, Biolegend, San Diego, CA, USA, and eBioscience, San Diego, CA, USA.

    Techniques: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Fluorescence

    Vav Cre GCS f/f showed a significant reduction of the invariant natural killer T (iNKT) cell population. (A) In thymi, spleens, and livers of 8-week-old wild-type (WT), Vav Cre GCS f/f and CD1d –/– mice, frequencies and absolute numbers of iNKT cells were measured by flow cytometry using PBS57-loaded CD1d tetramers and anti-CD44 antibodies. In spleens and livers, CD19 + cells were gated out. In Vav Cre GCS f/f mice, iNKT cells frequencies and numbers were significantly reduced in all three organs. CD1d-deficient mice served as negative controls. N = 10–13/group. (B) Thymic development of iNKT cells was investigated in 8-week-old mice. Antibodies against NK1.1 and CD44 were used to subdivide the developmental stages in immature (CD44 − /NK1.1 − ), semi-mature (CD44 + /NK1.1 − ), and mature (CD44 + /NK1.1 + ). Analyses were gated on iNKT cells defined as CD3 + /PBS57-CD1d + thymocytes. Shown are relative and absolute numbers (left and right panels, respectively) of iNKT cells with the corresponding phenotype. No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice in terms of relative numbers (i.e., distribution among the three stages). The significant reduction in absolute numbers reflected the overall diminished iNKT cell population in Vav Cre GCS f/f mice. N = 16/group in the left panel and 10/group in the right panel, respectively. (C) Usage of TCRVβ-chains by splenic iNKT cells was investigated in 8-week-old mice. Analyses were gated on CD19 − /PBS57-CD1d + /CD44 + splenocytes. Shown are relative and absolute numbers (left and right panels, respectively) of iNKT cells expressing the corresponding TCRVβ-chain. No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice in terms of relative numbers (i.e., distribution among the three TCRVβ-chains). The reduction in the absolute numbers reflected the diminished iNKT cell population in Vav Cre GCS f/f mice. N = 9/group in the left panel and 6/group in the right panel, respectively. (D,E) Proliferation and apoptosis of thymic iNKT cells were measured in 8-week-old mice using BrdU incorporation and Annexin V staining, respectively. In Vav Cre GCS f/f mice, iNKT cells (CD3 + /PBS57-CD1d + thymocytes) showed a significantly reduced proliferation and an increased apoptosis as compared to WT controls. By contrast, conventional thymocytes were unaffected. N = 5/group. Bars represent means ± SEM; * p

    Journal: Frontiers in Immunology

    Article Title: Glucosylceramide Synthase Is Involved in Development of Invariant Natural Killer T Cells

    doi: 10.3389/fimmu.2017.00848

    Figure Lengend Snippet: Vav Cre GCS f/f showed a significant reduction of the invariant natural killer T (iNKT) cell population. (A) In thymi, spleens, and livers of 8-week-old wild-type (WT), Vav Cre GCS f/f and CD1d –/– mice, frequencies and absolute numbers of iNKT cells were measured by flow cytometry using PBS57-loaded CD1d tetramers and anti-CD44 antibodies. In spleens and livers, CD19 + cells were gated out. In Vav Cre GCS f/f mice, iNKT cells frequencies and numbers were significantly reduced in all three organs. CD1d-deficient mice served as negative controls. N = 10–13/group. (B) Thymic development of iNKT cells was investigated in 8-week-old mice. Antibodies against NK1.1 and CD44 were used to subdivide the developmental stages in immature (CD44 − /NK1.1 − ), semi-mature (CD44 + /NK1.1 − ), and mature (CD44 + /NK1.1 + ). Analyses were gated on iNKT cells defined as CD3 + /PBS57-CD1d + thymocytes. Shown are relative and absolute numbers (left and right panels, respectively) of iNKT cells with the corresponding phenotype. No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice in terms of relative numbers (i.e., distribution among the three stages). The significant reduction in absolute numbers reflected the overall diminished iNKT cell population in Vav Cre GCS f/f mice. N = 16/group in the left panel and 10/group in the right panel, respectively. (C) Usage of TCRVβ-chains by splenic iNKT cells was investigated in 8-week-old mice. Analyses were gated on CD19 − /PBS57-CD1d + /CD44 + splenocytes. Shown are relative and absolute numbers (left and right panels, respectively) of iNKT cells expressing the corresponding TCRVβ-chain. No statistically significant differences could be observed between WT and Vav Cre GCS f/f mice in terms of relative numbers (i.e., distribution among the three TCRVβ-chains). The reduction in the absolute numbers reflected the diminished iNKT cell population in Vav Cre GCS f/f mice. N = 9/group in the left panel and 6/group in the right panel, respectively. (D,E) Proliferation and apoptosis of thymic iNKT cells were measured in 8-week-old mice using BrdU incorporation and Annexin V staining, respectively. In Vav Cre GCS f/f mice, iNKT cells (CD3 + /PBS57-CD1d + thymocytes) showed a significantly reduced proliferation and an increased apoptosis as compared to WT controls. By contrast, conventional thymocytes were unaffected. N = 5/group. Bars represent means ± SEM; * p

    Article Snippet: The following monoclonal antibodies were used: anti-CD1d (clone: 1B1); anti-CD3ε (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD11c (HL3), anti-CD19 (MB19-1), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-CD150/SLAM (9D1), anti-Ly108 (13G3), anti-MHCII (M5/144.15.2), anti-NK1.1 (PK136), anti-TCR-Vβ2 (B20.6), anti-TCR-Vβ7 (TR310), and anti-TCR-Vβ8.1 and 8.2 (MR5-2) from BD, Heidelberg, Germany, Biolegend, San Diego, CA, USA, and eBioscience, San Diego, CA, USA.

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Expressing, BrdU Incorporation Assay, Staining