BrdU (Bromodeoxyuridine) Antibody Staining Protocol for Immunohistochemistry


Description: 5-Bromodeoxyuridine (BrdU) is an analogue of thymidine. BrdU can be introduced to proliferating cells which in turn incorporate BrdU into the DNA during S phase, prior to cell division. Immunocytochemical staining with NCL-BrdU is an accurate method for measuring cell proliferation and detects nucleated cells from different animal species which have incorporated BrdU in place of thymidine into their DNA.


Primary Antibody

Name: BrdU Antibody

Clone: 85-2C8, Mouse anti-BrdU

Supplier: Novocastra Labs

Catalog Number: NCL-BrdU

Dilution: 1:200 - 1:400 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, or acetone fixed frozen 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