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BrdU (Bromodeoxyuridine) Antibody Staining Protocol for Immunohistochemistry
Description: The ability to measure DNA synthesis or cell proliferation is important in cell biology research. The measurement of cell proliferation or DNA synthesis by determining the incorporation of [3H]-thymidine into cellular DNA has become a widely used assay. [3H]-thymidine incorporated into DNA is detected by autoradiography, and this assay is labor intensive and uses expensive and potentially hazardous material, alternative assays have been developed. 5-bromo2’-deoxy-uridine (BrdU) can be incorporated into DNA in place of thymindine. Monoclonal antibodies directed against BrdU have been developed. Cells, which have incorporated BrdU into DNA, can be quickly detected using a monoclonal antibody against BrdU and an enzyme- or fluorochrome-conjugated secondary antibody. The binding of the antibody is achieved by denaturation of the DNA. This is usually obtained by exposing the cells to acid, base or heat.
Primary Antibody
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Name: BrdU Antibody |
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Clone: Mouse anti-BrdU |
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Supplier: Roche Diagnostic |
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Catalog Number: 1-299-964 |
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Dilution: 1:100 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed. |
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Incubation Time/Temp: 60min/room temperature |
Antigen Retrieval
| Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102) |
| Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) or 2N HCl (see Note below for details) |
| Heat/Cool Temperature: 95-100 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Nuclear |
| Images: Search image |
Additional Information:
| Tissue Type: BrdU incorporated tissues |
| Fixation: Formalin-fixed paraffin sections |
| Positive Control: BrdU incorporated tissues |
| Negative Control: Omit primary antibody, isotype control, absorption control |
| Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary |
Notes:
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Denature DNA by incubating sections in 2N HCl for 30 minutes at 37 °C, and neutralize the acid by immersing sections in 0.1M borate buffer for 2x5 min.
2N
HCl:
10N HCl --------------------------- 20 ml
Distilled water -------------------- 80 ml
Mix well.
0.1M Borate
Buffer, pH 8.5:
Sodium borate (MW 381.4) ----- 3.8 g
Distilled water -------------------- 100 ml |
References: