BrdU, also known as 5-bromo-2-deoxyuridine, is a base analog of thymidine, with the thymine substituted by bromouracil. Because BrdU can replace thymidine, excessive exposure to the chemical can cause mutations. A base analog is a chemical that can substitute a normal nucleobase in nucleic acids. ... Deoxythymidine is a molecule (known as a nucleoside) that is formed when thymine is attached to a deoxyribose ring (also known as a deoxyribofuranose) via a β-N1-glycosidic bond. ... Thymine, also known as 5-methyluracil, is a pyrimidine nucleobase. ... 5-bromouracil (or 5-Bromo-2,4(1H,3H)-pyrimidinedione or 5-BrU) is a component of 5-bromo-2-deoxy-uridine. ... In biology, mutations are changes to the genetic material (usually DNA or RNA). ...

BrdU is a common chemical used in the detection of proliferating cells in living tissues. It works by substituting for thymidine during DNA replication (being a base analog of thymidine) and incorporating itself into the newly synthesized DNA (during S-phase). Antibodies specific for BrdU can then be used to detect the incorporated chemical (see immunohistochemistry), thus indicating cells that were actively replicating its DNA (proliferating). It is frequently used in conjunction with antibodies that reveal cell type in what are known as "double-labeling" experiments. DNA replication. ... For other uses, see DNA (disambiguation). ... Schematic of antibody binding to an antigen An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. ... Immunohistochemistry is the process of detection of antigens in tissue using antibodies. ...

BrdU Immunohistochemical Staining Protocol

Description: To measure DNA synthesis or cell proliferation, 5-bromo2’-deoxy-uridine (BrdU) can be incorporated into DNA in place of thymindine. Cells, which have incorporated BrdU into DNA, can be quickly detected using a monoclonal antibody against BrdU. The binding of the antibody is achieved by denaturation of the DNA. This is usually obtained by exposing the cells to acid, or heat.

Fixation: Formalin-fixed, paraffin embedded sections.

Positive Controls: BrdU incorporated tissues.

Solutions and Reagents:

Primary Antibody: Rat Anti-Human BrdU (Clone BU1/75, ICR1) (Accurate Chemical & Scientific, Cat# OBT0030). Optimal dilution 1:100. Species Reactivity: Human, mouse, rat (refer to antibody datasheet for more information).

Secondary Antibody: Rabbit Anti-Rat IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-4001). Optimal dilution 1:500.

Detection Reagent: HRP-Streptavidin (Vector Laboratories, Cat# SA-5004). Optimal dilution 1:500

Procedure:

1. Paraffin sections to distilled water (refer to general IHC protocols as needed). 2. Epitope Retrieval: Use Citrate Buffer Epitope Retrieval Method. Briefly, pre-heat steamer or water bath with staining dish containing Citrate Buffer (pH 6.0) until temperature reaches 95-100 °C. Immerse slides in the staining dish and place the lid loosely on the staining dish. Incubate for 20 minutes and turn off the steamer or water bath. Place the staining dish at room temperature and allow the slides to cool for 20 minutes. 3. Rinse sections in 2 changes of washing buffer, 2 minutes each. 4. Serum Blocking: incubate sections with normal rabbit serum blocking solution for 30 minutes to block non-specific binding of immunoglobulin. 5. Primary Antibody: incubate sections with Rat Anti-BrdU (Accurate Chemical & Scientific, Cat# OBT0030) diluted 1:100 in primary antibody dilution buffer for 1 hour at room temperature. 6. Rinse in washing buffer for 2x2min. 7. Peroxidase Blocking: incubate sections in peroxidase blocking solution for 10 minutes to block endogenous peroxidase activity. 8. Rinse in washing buffer for 3x2 min. 9. Secondary Antibody: incubate sections with biotinylated Rabbit Anti-Rat IgG diluted in secondary antibody dilution buffer for 30 minutes at room temperature. 10. Rinse in washing buffer for 3x2min. 11. Detection: incubate sections with HRP-Streptavidin diluted in HRP-streptavidin dilution buffer for 30 minutes at room temperature. 12. Rinse in washing buffer for 3x2min. 13. Chromogen/Substrate: incubate sections in DAB peroxidase substrate solution for 5-10 minutes. 14. Rinse in distilled water briefly. 15. Counterstain with Gill's hematoxylin solution or Mayer's hematoxylin solution if desired. 16. Rinse in running tap water for 5 minutes. 17. Dehydrate through 95% ethanol for 2 minutes, 100% ethanol for 2x3min. 18. Clear in xylene for 2x3min. 19. Coverslip with permanent mounting medium.