Anti-BrdU抗体[BU1/75 (ICR1)] (ab6326)

概述

  • 产品名称Anti-BrdU抗体[BU1/75 (ICR1)]
    参阅全部 BrdU 一抗
  • 描述
    大鼠单克隆抗体[BU1/75 (ICR1)] to BrdU
  • 特异性This antibody reacts with BrdU in single stranded DNA, BrdU attached to a protein carrier or free BrdU. It detects nucleated cells in S-Phase which have had BrdU incorporated into their DNA. Also reacts with chlorodeoxyuridine but with reduced staining. The antibody does not react with thymidine. The antibody does not cross react with IdU.
  • 经测试应用适用于: ICC/IF, IHC-FoFr, IHC-P, IHC (PFA fixed), IHC-P, ICC, IHC-Fr, Flow Cyt, IHC-FrFlmore details
  • 种属反应性
    Not applicable.
  • 免疫原

    The details of the immunogen for this antibody are not available.

  • 常规说明The antibody recognises single stranded DNA so the DNA needs to be unraveled first. This can be done with DNAse, although this doesn't give the best results. Depending on the assay, acid denaturation with 2M HCL or heat denaturation are the most successful. Please note this step is critical in any assay with this antibody and is the area that should be modified to optimise results. Detailed BrdU protocol is available in "Neuroscience protocols" on our "Protocol and troubleshooting tips" webpage (www.abcam.com/protocols).

性能

应用

Our Abpromise guarantee covers the use of ab6326 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
ICC/IF 1/250.
IHC-FoFr 1/40.
IHC-P 1/40.
IHC (PFA fixed) 1/40.
IHC-P Use at an assay dependent concentration. PubMed: 26192438
ICC Use at an assay dependent concentration.
IHC-Fr 1/40 - 1/200. PubMed: 16670699In addition, found to work at 1/400. For PFA fixed tissue use at 1/40, from PMID 16373695.
Flow Cyt 1/25 - 1/200.

ab18450 - Rat monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

IHC-FrFl Use at an assay dependent concentration.

靶标

  • 相关性The immunocytochemical detection of bromodeoxyuridine (BrdU) incorporated into DNA is a powerful tool to study the cytokinetics of normal and neoplastic cells. In vitro or in vivo labeling of tumor cells with the thymidine analogue BrdU and the subsequent detection of incorporated BrdU with specific anti-BrdU monoclonal antibodies is an accurate and comprehensive method to quantitate the degree of DNA-synthesis. BrdU is incorporated into the newly synthezised DNA of S-phase cells may provide an estimate for the fraction of cells in S-phase. Also dynamic proliferative information such as the S-phase transit rate and the potential doubling time can be obtained, by means of bivariate BrdU/DNA flow cytometric analysis.
  • 细胞定位Nuclear
  • 别名
    • Bromodeoxyuridine antibody
    • BUdr antibody

Anti-BrdU antibody [BU1/75 (ICR1)] 图像

  • ab6326 stained in Hela cells. Untreated and BrdU treated (10uM for 24 hours) cells were fixed with 100% methanol (5 min) and then subjected to acid hydrolysis using 2M HCL in 0.1% PBS-Tween for 30 minutes at room temperature to denature the DNA. They were then incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab6326 at 5µg/ml and ab7291 (Mouse monoclonal to alpha tubulin) at 1ug/ml overnight at +4°C. The secondary antibodies were ab1501656 (colored green) used at 2 ug/ml and ab151020 (pseudo-colored red) used at 1/1000 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • ICC/IF image of ab6326 stained HeLa cells, both BrdU treated (left image) and normal cells (right image). The cells were 100% methanol fixed (5 min) and then subjected to acid hydrolysis using 2M HCL in 0.1% PBS-Tween for 30 minutes at room temperature to denature the DNA. They were then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6326, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab98420, DyLight® 488 goat anti-rat IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. Positive staining can be seen in the BrdU treated cells, but not in the normal cells, demonstrating specificity for BrdU.

  • Dot plot showing BrdU-treated cells stained with ab6326. Cells were incubated with 10 µM BrdU for 30 minutes prior to being harvested, washed twice in 1x PBS and fixed in 70% ethanol (4°C, added drop-wise) for at least 30 minutes on ice. Once fixed, pellets were acid denatured with 2M HCl for 30 minutes at 22ºC and then neutralised with borate buffer (0.1M, pH8.5).

    Samples were washed and incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab6326, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rat IgG (H&L) (preadsorbed) (ab150165) at 1/2000 dilution for 30 min at 22ºC.

    7-AAD was added to cells 20 min prior to data acquisition.

    Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) with 530/30 and 685/35 bandpass filters.

  • ab6326 staining BrdU in HeLa cells by Flow Cytometry. Cells were incubated with 10 µM BrdU for 30 minutes prior to being harvested with 1X trypsin-EDTA, washed twice in PBS containing 1% BSA, and fixed in 70% ethanol (added drop-wise) for at least 30 minutes on ice. Once fixed, pellets were acid denatured with HCl/Triton X-100 for 30 minutes at room temperature and then neutralised with sodium tetraborate.
    Pelleted cells were re-suspended in Tween/BSA/PBS to which primary antibody was then added (0.1 µg in 0.5% Tween 20 (v/v) plus 1% BSA in PBSA) and incubated for 30 minutes at room temperature. Secondary Alexa Fluor®488-conjugated Goat anti-Rat IgG (H+L) was used at 1/500 and incubated for 30 minutes at room temperature in the dark. Cells were pelleted once more and resuspended in PBS containing 5 µg/mL propidium iodide.
    Gating Strategy: Based on forward and side scatter, cells were gated into the region used for analysis. This was done by applying a large circle to a

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  • IHC image of ab6326 staining in a formalin-fixed, paraffin-embedded rat small intestine BrdU tissue section. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4°C with ab6326 at 1 ug/ml. A goat anti-rat biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. The section was counterstained with haematoxylin and mounted with DPX.

     

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • ab6326 staining BrdU in mouse brain (dentate gyrus) tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were perfused with 1X PBS followed by 4% paraformaldehyde and then cryopreserved in 20-30% sucrose. 20-25 µm sections were permeablized with 1% Triton X-100 + 0.5% Tween 20 in 1X PBS. Sections were treated with 1 N HCL for 10 min followed by 2 N HCL for 10 min at RT and then 20 min at 37°C. Then sections were incubated with borate for pH correction and permeablized with 1 X TBS and blocked with 3-5% donkey serum.  Samples were incubated with the primary antibody (1/1000 in 1X PBS + 0.1% Tween 20) at 4°C overnight. ab175475, an Alexa Fluor® 568-conjugated donkey anti-rabbit IgG (H+L) polyclonal (1/250) was used as the secondary antibody.

    Green - DCX.
    Red - BrdU.
    Blue - NeuN.

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  • ab6326 staining BrdU in mouse brain tissue sections by IHC-Fr (paraformaldehyde-fixed frozen sections). Tissue samples were fixed with paraformaldehyde; permeabilized win 0.3% Triton X-100 and blocked with 5% Serum for 2 hours at 4°C. Before permeabilization samples were pretreated with 2N HCl at RT for 30 min and washed 3 times. The sample was incubated with primary antibody (1/200) at 4°C for 12 hours. An Alexa Fluor® 488-conjugated Goat polyclonal to rat IgG (1/500) was used as secondary antibody. BrdU staining shown in green and NewN staining showin in red.

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  • ab6326 staining cultured cells of rat brain tissue by ICC.  The sample was PFA fixed and permeabilized in 1M HCl prior to blocking with 5% serum for 1 hour at 25°C.  The primary antibody was diluted 1/500 and incubated with the sample for 16 hours at 25°C.  A biotinylated rabbit anti-rat IgG antibody, diluted 1/200, was used as the secondary.

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Anti-BrdU antibody [BU1/75 (ICR1)] (ab6326)参考文献

This product has been referenced in:
  • You J  et al. DDX59 promotes DNA replication in lung adenocarcinoma. Cell Death Discov 3:16095 (2017). ICC/IF . Read more (PubMed: 28090355) »
  • Wu R  et al. H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication. Nucleic Acids Res 45:169-180 (2017). Read more (PubMed: 27679476) »

See all 585 Publications for this product

Product Wall

Application Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample Mouse Tissue sections (Brain)
Specification Brain
Username

Mr. Musaad Alshammari

Verified customer

提交于 Jan 11 2016

Application Immunohistochemistry free floating
Sample Mouse Tissue sections (Brain)
Specification Brain
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Abcam user community

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提交于 Jan 14 2016

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Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (Human cerebral organoids)
Specification Human cerebral organoids
Blocking step Serum as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: 4°C
Fixative Paraformaldehyde
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Verified customer

提交于 Jul 13 2017

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Application Immunohistochemistry free floating
Sample Mouse Tissue sections (Brain sections)
Specification Brain sections
Username

Yacine Tensaouti

Verified customer

提交于 Jun 14 2017

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Application Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample Mouse Tissue sections (BRAIN)
Antigen retrieval step None
Permeabilization Yes - 4% paraform
Specification BRAIN
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Fixative Paraformaldehyde
Username

Kyungjoo Seong

Verified customer

提交于 Dec 21 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (lung)
Permeabilization Yes - 2N HCl
Specification lung
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 20°C
Fixative Paraformaldehyde
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Abcam user community

Verified customer

提交于 Dec 14 2016

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Human Tissue sections (xenograft grown on nude mouse)
Antigen retrieval step Other
Permeabilization No
Specification xenograft grown on nude mouse
Blocking step BSA as blocking agent for 20 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative Formaldehyde
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Abcam user community

Verified customer

提交于 Nov 21 2016

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (U2OS)
Permeabilization Yes - Np40
Specification U2OS
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative Formaldehyde
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Abcam user community

Verified customer

提交于 Aug 17 2016

Application Immunocytochemistry/ Immunofluorescence
Sample Monkey Cell (Kidney)
Permeabilization Yes - 0.1% triton x-100
Specification Kidney
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

提交于 Jul 29 2016

Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (Brain)
Permeabilization No
Specification Brain
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

提交于 Mar 04 2016

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