Overview

  • Product name
    Anti-BrdU antibody [IIB5]
    See all BrdU primary antibodies
  • Description
    Mouse monoclonal [IIB5] to BrdU
  • Host species
    Mouse
  • Specificity
    BrdU is a thymidine analogue and when offered to proliferating cells it is incroporated into reduplicating cells. The antibody is specific for DNA in which BrdU has been incorporated. In immunoassays this antibody reacts strongly with free or carrier-protein coupled BrdU but not with other nucleosides. In immuncytochemistry the antibody only recognizes BrdU in denaturated (single stranded) DNA. The BrdU antibody is 100% crossreactive with Iodo-Deoxy-Uridine (IrdU). Therfore, IdU instead of BrdU can be used in studies.
  • Tested applications
    Suitable for: IHC-FoFr, ICC/IF, Flow Cyt, IHC-Fr, ICC, IHC-Pmore details
  • Immunogen

    Chemical/ Small Molecule BrdU conjugated to BSA.

  • Positive control
    • Bromodeoxyuridine labeled cells.
  • General notes

    The following product is available as purified antibody (purified by affinity chromatography) together with several conjugated versions:

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

Properties

Applications

Our Abpromise guarantee covers the use of ab8152 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-FoFr Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IHC-Fr 1/5 - 1/10.

Dilute antibody in 0.15 M phosphate buffered saline with 1% BSA and 1% Na-azide.

ICC Use at an assay dependent concentration.
IHC-P 1/5 - 1/10. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Enzymatic antigen retrieval with proteases can also be used.

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

  • ab8152 (1/100) staining BrdU in HeLa cells (green). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X100/ PBS and counterstained with DAPI in order to highlight the nucleus (red). For further esperimental details please see Abreview.

    See Abreview

  • Immunohistochemical analysis of rat brain tissue, staining BrdU with ab8152.

    Tissue was fixed with paraformaldehyde and blocked with 5% serum for 1 hour at 25°C; antigen retrieval was by heat mediation in citrate buffer (pH 6). Samples were incubated with primary antibody (1/20 in diluent) for 18 hours at 25°C. A Cy3®-conjugated donkey anti-mouse polyclonal IgG was used as the secondary antibody.

    See Abreview

  • Cells were pulse labeled with 10 mM BrdU for 30 min, rinsed twice in prewarmed PBS, and chased in prewarmed culture medium, supplemented with 5 mM deoxythymidine. Incorporated BrdU was detected after ethanol fixation of the cells, which were than rinsed once in PBS and resuspended in 2 ml of 0.4 mg/ml pepsin in 0.1 N HCl. After 30 min at room temperature cells were pelleted, resuspended in 2 N HCl, and incubated for another 30 min at 37°C. Cells were rinsed in 0.1 M borate buffer, pH 8.5, and PBS/BSA (1 mg/ml BSA in PBS). Appropriately diluted mouse anti-BrdU antibody (clone IIB5) was added to the cell pellet, resuspended in 100 micro liters PBS/BSA. After incubation for 1 h at room temperature, the cells were rinsed twice in PBS/BSA. For visualization, FITC-conjugated Fab2 fragments of rabbit anti-mouse Ig antibody were added in a 1/10 dilution. After incubation for 45 min at room temperature samples were rinsed twice in PBS/BSA and the cells were finally resuspended in 0.5 ml cold PBS supplemented with 100 microgram/ml RNAse and 20 µg/ml propidium iodide. The samples were allowed to stand for 15 min on ice in the dark before flow cytometric analysis. In the negative control the primary antibody was omitted.

References

This product has been referenced in:
  • Gao J  et al. In vitro investigation of the mechanism underlying the effect of ginsenoside on the proliferation and differentiation of neural stem cells subjected to oxygen-glucose deprivation/reperfusion. Int J Mol Med 41:353-363 (2018). Read more (PubMed: 29138802) »
  • Wang X  et al. Downregulation of YAP inhibits proliferation, invasion and increases cisplatin sensitivity in human hepatocellular carcinoma cells. Oncol Lett 16:585-593 (2018). Read more (PubMed: 29928445) »
See all 23 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rabbit Tissue sections (Intestine)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 2M HCl
Permeabilization
Yes - Triton X-100
Specification
Intestine
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Formaldehyde

Dr. Egi Kardia

Verified customer

Submitted Sep 21 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Rabbit Cell (Primary airway epithelial cells)
Permeabilization
No
Specification
Primary airway epithelial cells
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
70% Ethanol

Dr. Egi Kardia

Verified customer

Submitted Sep 06 2018

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C
Sample
Human Cell (OCI ly1)
Specification
OCI ly1
Permeabilization
Yes - 0.25% Triton X-100 in PBS
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Nov 07 2014

Answer

Thank you for your inquiry and for rating your experience with us.

I just wanted to follow up on your query with some additional information.

For ab8039, this antibody can be used for detection of BrdU and BrU. We have not tested it regarding other chemicals of this family.

For ab8152, this antibody is specific for DNA in which BrdU has been incorporated. In immunoassays this antibody reacts strongly with free or carrier-protein coupled BrdU but not with other nucleosides. In immuncytochemistry the antibody only recognizes BrdU in denaturated (single stranded) DNA. The BrdU antibody is 100% crossreactive with Iodo-Deoxy-Uridine (IrdU). Therfore, IdU instead of BrdU can be used in studies.

I hope this information helps. Please contact us with any other questions.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (brain)
Specification
brain
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 07 2012

Answer

Thank you for contacting us.

For floating sections, please check thefollowing protocol on IHC World website:
http://www.ihcworld.com/_protocols/epitope_retrieval/free_floating_sections.htm
This protocol is for paraffin-embedded as well as frozen sections.

As for ensuring the paraffin sections stick to your slides:
Different sections and tissues stick with differetn strength to the slides.
It might be advisable to use charged slides, as well as to bake your sections onto the slides by exposing them to heat (e.g. heating plate or oven) up to 60 degree Celsius.The purpose is to remove miscroscopic amounts of water between the sections and the slide.
Subsequently the paraffin would need to removed according to a standard IHC-P protocol. Then you would proceed with the antigen retrieval step.

Also, please check this information on the IHC World website for further advice.
http://www.ihcworld.com/_faq/histology-faq/section/s1.htm

Read More

Answer

The slides need to be immersed, for the retrieval to be effective. Yes, the sections could come off ifthey are kept too long in the retrieval solution. Should they come off, you would need to reduce the time they are exposed to the retrieval buffer.

Are the floating sections paraffin-embedded or frozen? I have found the following protocol on IHC World website which should help you further:
http://www.ihcworld.com/_protocols/epitope_retrieval/free_floating_sections.htm

Read More

Answer

I have checked with the lab:
Antigen retrieval can be done with HIER using citrate buffer.

Please see below for the protocol:

High Temperature antigen Unmasking Technique using Sodium Citrate Buffer for Immunohistochemical Demonstration on Paraffin Sections

1. Cut and mount sections on slides coated with Vectabond or APES (3-aminopropyltriethoxysilane).
2. Deparaffinize sections and rehydrate to distilled water.
3. Bring 1600ml 0.01M sodium citrate buffer (pH 6.0) to the boil in a Prestige stainless steel pressure cooker, using a hot plate. Cover but do not lock lid.
4. Position slides into metal staining racks and lower into pressure cooker ensuring slides are well immersed in citrate buffer. Lock lid. The small valve will rise.
5. When the pressure indicator valve (the large one) has risen after about 4 minutes, incubate sections for 1 minute.
6. Remove pressure cooker from heat source and run under cold water with lid on. When the small valve sinks open lid and remove slides and place immediately into distilled water. DO NOT OPEN LID UNTIL THE SMALL VALVE SINKS.
7. Wash sections in TBS buffer (pH 7.6) for 1 x 5 minutes.
8. Place sections in 1.5% hydrogen peroxide/methanol for 10 minutes.
9. Wash sections in distilled water for 2 x 5 minutes, then wash sections in TBS buffer for 2 x 5 minutes.
10. Place sections in normal serum for 20 minutes.
11. Cover sections with primary antibody. ( The optimal dilution of the antibody, incubation time and incubation temperature should be determined by the individual laboratory).
12. Wash in TBS buffer for 2 x 5 minutes.
13. Incubate sections in secondary antibody for 30 minutes.
14. Wash in TBS buffer for 2 x 5 minutes.
15. Incubate slides in ABComplex for 30 minutes.
16. Wash in TBS buffer for 2 x 5 minutes.
17. Incubate slides in DAB.
18. Wash in water for 2 x 5 minutes.
19. Counterstain with haematoxylin (if required), dehydrate, coverslip and mount.


To avoid sections becoming detached, sections should be mounted on Vectabond or APES covered slides, then dried at 37oC overnight followed by drying at 56oC for 60 minutes.

Read More

Answer

Thank you for contacting us with your question. We unfortunately do not have any data regarding the minimum length of ssDNA required for antibody staining on adherent cells.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
African Green Monkey Cell (Oligodendrocytes, astrocytes (mixed glial culture))
Specification
Oligodendrocytes, astrocytes (mixed glial culture)
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.3% Triton-PBS for 30 minutes followed by DNAse treatment for 90 minutes.

Abcam user community

Verified customer

Submitted Oct 28 2010

1-10 of 12 Abreviews or Q&A

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

Sign up