Overview

  • Product name
    Anti-BrdU antibody (Biotin)
    See all BrdU primary antibodies
  • Description
    Sheep polyclonal to BrdU (Biotin)
  • Host species
    Sheep
  • Conjugation
    Biotin
  • Specificity
    The antibody was tested using immunoprecipitation against 5-Methyl Cytosine (5-MeC) and bromo-deoxyuridine (BrdU) or control (no antigen) and assayed by A-405 spectrophotometry. At a concentration of 25 µg/mL, this product demonstrates 8 fold higher reactivity against BrdU than 5-MeC. This product titers out at 50 ng/mL. For best results, use the product at a concentration of 25 to 100 µg/mL. Nearly complete immunoprecipitation was obtained at a concentration of 100 to 500 µg/mL.
  • Tested applications
    Suitable for: IHC-P, IHC-Fr, IP, ICC/IF, ELISA, IHC-FoFrmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Bromodeoxyuridine coupled to keyhole limpet hemocyanin (KLH)

Properties

Applications

Our Abpromise guarantee covers the use of ab2284 in the following tested applications.

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

Application Abreviews Notes
IHC-P Use at an assay dependent dilution.
IHC-Fr Use at an assay dependent dilution. Fixation in cold methanol for 30 minutes followed by immersion in 7 x 10-3 N NaOH for 10-15 seconds allows BrdU staining with the simultaneous detection of nuclear cytoplasmic and membrane assigns as well as preservation of morphological detail.
IP Use at an assay dependent dilution.
ICC/IF Use at an assay dependent dilution.
ELISA Use at an assay dependent dilution. dilute from 1/500 to 1/40,000 against 1mg/mL BrdU analyte.
IHC-FoFr 1/2000. 1/2000 (see Abreview).

Target

  • Relevance
    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.
  • Cellular localization
    Nuclear
  • Alternative names
    • Bromodeoxyuridine antibody
    • BUdr antibody

Images

  • ab2284 staining BrdU in mouse intestine tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% H2O2 in methanol for 12 minutes; antigen retrieval was by heat mediation in 10mM citrate, pH6. Samples were incubated with primary antibody (1/100) for 24 hours at 4°C.

    See Abreview

  • ab2284 at 1/250 staining primary E12 mouse cortex cells by ICC/IF. The cells were paraformaldehyde fixed, blocked with serum and then incubated with the antibody for 24 hours. Streptavidin conjugated to Alexa-Fluor ®  488 was used as the secondary. The image shows BrdU staining with nuclei counterstained with DAPI.

    See Abreview

  • ab2284 staining BrdU in Mouse skin tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with acetone and blocked with 10% serum for 30 minutes. Samples were incubated with primary antibody (1/50 in PBS) for 12 hours at 4°C. A Streptavidin Alexa Fluor® 488-conjugated Goat polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • ab2284 at 1/2000 dilution staining mouse free floating brain slices by Immunohistochemistry (Formalin/PFA fixed sections). The mice were treated with 100mg/kg BrdU 2 hours before fixation. Free floating 40µm vibratome sections were obtained from paraformaldehyde fixed brains, these were incubated with the antibody for 24 hours. A streptavidin-HRP complex and DAB were used for detection. The image depicts the subventricular zone.

    See Abreview

References

This product has been referenced in:
  • Razumilava N  et al. Hedgehog Signaling Modulates Interleukin-33-Dependent Extrahepatic Bile Duct Cell Proliferation in Mice. Hepatol Commun 3:277-292 (2019). Read more (PubMed: 30766964) »
  • Gruffaz M  et al. Repurposing Cytarabine for Treating Primary Effusion Lymphoma by Targeting Kaposi's Sarcoma-Associated Herpesvirus Latent and Lytic Replications. MBio 9:N/A (2018). Read more (PubMed: 29739902) »
See all 32 Publications for this product

Customer reviews and Q&As

11-13 of 13 Abreviews or Q&A

Answer

I would recommend the following steps: -Deparaffinize and rehydrate sections -Block endogenous peroxidase activity with 3% H2O2 for 10 minutes at 37 °C -Perform DNA denaturation -Perform antigen retrieval -Perform permeabilisation (I prefer adding 0.3% tritonx100 in the blocking and antibody buffers) -Block in serum -Add Primary antibody ( a customer reported that 1:2000 worked well, so I would indeed try 1:500-1:8000)(overnight incubation at 4C works best) -Continue Immunostaining procedure

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Question
Answer

Thanks for your recent phone call. I enclose below the protocol that we talked about, which was used for a number of BrDU antibodies and gave great staining in my old lab (By Dan Webber): BrdU protocol Bromodeoxyuridine (BrdU) uses nucleotide substitution to replace thymidine with uridine in the DNA structure of dividing cells both in vitro and in vivo (Gage, 2000). BrdU has been utilised in a number of in vitro and in vivo studies to label a variety of cellular sources from human olfactory epithelium (Hahn et al., 2005) and neural progenitors (Nieoullon et al., 2005) to neural stem cells (Chu et al., 2004). BrdU antibody from Abcam Identification of the labelled cells can be applied both in vitro and in vivo to produce a nuclear staining pattern. Protocol Tissue and cells are fixed with 4% paraformaldehyde in vitro for 30 mins at 4ºC, in vivo post fix for two hours at room temperature (r.t.) Following fixation, wash in 0.1M Phosphate Buffered Saline (PBS) (pH 7.4) with 1% TritonX100 (3x 5 mins) Incubate in HCl (1N) for 10 mins on ice to break open the DNA structure of the labelled cells This is followed by HCl (2N) for 10 mins at r.t before moving them to an incubator for 20 mins at 37°C Immediately after the acid washes, Borate buffer (0.1M) is added to buffer the cells for 12 mins at r.t. Samples are then washed in wash in 0.1M PBS (pH 7.4) with 1% TritonX100 (3x 5 mins) at r.t. Incubate in 0.1M PBS (pH 7.4) + 1% TritonX100 + Glycine (1M) + 5% normal goat serum (1hr) prior to incubating overnight (r.t) with anti-BrdU or a combination of anti-BrdU and other antibodies. Following the incubation overnight wash in 0.1M PBS (pH 7.4) with 1% TritonX100 (3x 5min) Samples can then be treated with a variety of secondary antibodies to visualise the anti-BrdU labelled cells (Fig 1 and Fig 2) including HRP conjugated secondaries with diaminobenzidine(DAB) or fluorescent conjugated antibodies such as AlexaFlour 488 or 533. Tissue processing tips In vivo and in vitro samples can be processed in this manner: Gelatin embedded tissue (cut between 30-50µm) OCT embedded tissue (cut between 10-30µm) Tissue culture cells NB: The acid steps haven’t shown any adverse affect on the labelling of any other antigens tested so far in double labelling. Incubation times haven’t needed to be altered with thickness of section and everything is treated in the same manner You can also access this through this link: https://www.abcam.com/index.html?pageconfig=resource&rid=10203&pid=7 I have looked at the datasheet of ab1893 (the non conjugated version of ab2284) and found that Dan has used this antibody with his BrDU protocol, so it should work well for you too (you'll have to alter the secondary step since you have an antibody already biotinylated). A review of ab2284 by another user also reported that Protease V was not used and the antibody still gave good results (you can view the review on the online datasheet). I hope this will help you, please do not hesitate to contact me if I can be of further assistance,

Read More
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Cell (free floating Brain slices)
Specification
free floating Brain slices
Fixative
Paraformaldehyde
Antigen retrieval step
None
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10

Dr. Christoph Schwarzer

Verified customer

Submitted Nov 03 2006

11-13 of 13 Abreviews or Q&A

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