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BrdU Antibody Staining Protocol for Immunohistochemistry
Description: Bromodeoxyuridine (BrdU), an analog of thymidine (derivative of uridine), is a uridine derivative that can be incorporated specifically into DNA in place of thymidine. Cells can be pulse-labeled with BrdU, and those cells that are synthesizing DNA (in S-phase of the cell cycle) will incorporate BrdU into the DNA. Anti-BrdU can then be used to identify cells that undergo DNA synthesis during exposure to BrdU. The proportion of cells in S-phase of the cell cycle can be determined either by fluorescence microscopy or by flow cytometric analysis. Anti-BrdU identifies BrdU (but not thymidine) in single-stranded DNA, free BrdU, or BrdU coupled to a protein carrier. The antibody also reacts with iodouridine.
Primary Antibody
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Name: BrdU Antibody |
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Clone: Mouse anti-BrdU |
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Supplier: Immunocytometry System |
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Catalog Number: 347580 |
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Dilution: 1:50 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: