Overview

  • Product nameAnti-BrdU [BU1/75 (ICR1)] antibodySee all BrdU primary antibodies ...
  • Description
    Rat monoclonal [BU1/75 (ICR1)] to BrdU
  • SpecificityThis 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.
  • Tested applicationsICC/IF, IHC-FoFr, IHC-P, IHC (PFA fixed), ICC, IHC-Fr, Flow Cyt, IHC-FrFl, IHC - Wholemount more details
  • Species reactivity
    Not applicable.
  • Immunogen

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

  • General notesThe 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).

Properties

Applications

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.

Application Notes
ICC/IF 1/250.
IHC-FoFr 1/40.
IHC-P 1/40.
IHC (PFA fixed) 1/40.
ICC Use at an assay dependent dilution.
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 1/40.
IHC-FrFl Use at an assay dependent concentration.
IHC - Wholemount Use at an assay dependent concentration.

Target

  • RelevanceThe 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.
  • Cellular localizationNuclear
  • Alternative names
    • Bromodeoxyuridine antibody
    • BUdr antibody

Anti-BrdU [BU1/75 (ICR1)] antibody images

  • 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.

  • Paraffin-embedded sections of hippocampus dentate gyrus stained for  BrdU-positive cells. The method is an indirect immunohistochemical technique: the primary antibody is ab6326 diluted 1/100 in PBS and the secondary antibody is an Alexa 488-conjugated anti-rat made in goat diluted 1/200.
  • ab6326 used in double labeling on mouse sciatic nerve tissue (5µm) sections. This picture was kindly submitted by Dr Ulrich Hengst as part of his review on this product.

  • ab6326 at 1/200 staining mouse E14.5 embryonic telencephalon tissue sections by IHC-P. The tissue was formaldehyde fixed and a heat mediated antigen retireval step was performed. The tissue was then stained with the antibody for 12 hours. A Texas Red conjugated donkey anti-rat antibody was used as the secondary.

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  • ab6326 staining mouse adult epidermis tissue sections by IHC-P.  Sections were PFA fixed and subjected to heat mediated antigen retrieval in citrate buffer prior to blocking in 10% serum for 1 hr at RT.  The primary antibody was diluted 1/200 and incubated with the sample for 24 hrs at 4°C.  A biotinylated goat anti-rat antibody was used as the secondary followed by a streptavidin-Cy3®.

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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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  • 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 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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References for Anti-BrdU [BU1/75 (ICR1)] antibody (ab6326)

This product has been referenced in:
  • Renaud E & Rosselli F FANC pathway promotes UV-induced stalled replication forks recovery by acting both upstream and downstream Pol? and Rev1. PLoS One 8:e53693 (2013). ICC/IF . Read more (PubMed: 23365640) »
  • Jászai J  et al. Spatial distribution of prominin-1 (CD133)-positive cells within germinative zones of the vertebrate brain. PLoS One 8:e63457 (2013). Read more (PubMed: 23723983) »

See all 168 Publications for this product

Product Wall

Thank you for your response. I am very pleased to hear that the vials have arrived in good conditions.

These two vials (http://ops.adminsite.com/admin/stock/stock_list.cfm?intAbID=6326&intStockID=1343901&fMain=1&fViewExpired=1&...

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Thank you for your call today and for letting us know about the trouble with these antibodies.

Please keep me updated about the results with these antibodies after trying the alterations that we discussed. Here is the article that I mentioned...

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Application Immunohistochemistry (Frozen sections)
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Sample Mouse Tissue sections (hippocampus)
Specification hippocampus
Permeabilization Yes - 2N HCl
Fixative Paraformaldehyde
Username

Miss. Sung-Eun WANG

Verified customer

Submitted Apr 17 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citrate pH 6.0 6-23 min microwave or 30 min. 60¯C 2N HCl followed by 25 min. 0.1M Sodium Borate Buffer pH 8.5 RT
Sample Mouse Tissue sections (mouse brain)
Specification mouse brain
Permeabilization Yes - Triton X-100
Fixative Paraformaldehyde
Username

Miss. Verena Pfeiffer

Verified customer

Submitted Apr 02 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Sample Human Cell (MCF10A/ U2OS)
Specification MCF10A/ U2OS
Permeabilization No
Fixative Formaldehyde
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Abcam user community

Verified customer

Submitted Nov 25 2013

The antibody is applicable in assays that detect BrdU. For detection of BrdU in whole cultured cells (as opposed to lysates of the cells), the cells will need to be fixed and permeabilized to facilitate antibody access to the BrdU. So, it will not immu...

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Application IHC - Wholemount
Sample Mouse Tissue (hippocampus)
Specification hippocampus
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Abcam user community

Verified customer

Submitted Sep 04 2013

Application IHC - Wholemount
Sample Mouse Tissue (hippocampus)
Specification hippocampus
Username

Abcam user community

Verified customer

Submitted Aug 28 2013

Application Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample Mouse Tissue sections (brain)
Specification brain
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: 50%foramide
Permeabilization Yes - 2N HCl
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Apr 16 2013

Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (Brain)
Specification Brain
Fixative Formaldehyde
Permeabilization No
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Username

Abcam user community

Verified customer

Submitted Apr 12 2013

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"