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

Name: BrdU Antibody

Clone: Mouse anti-BrdU

Supplier: Immunocytometry System

Catalog Number: 347580

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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


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

Mix to dissolve and adjust pH to 8.5