BrdU Protocol

BrdU (Bromodeoxyuridine / 5-bromo-2'-deoxyuridine) is an analog of the nucleoside thymidine normally used in the BrdU assay to identify proliferating cells. BrdU labeling can be performed in vitro or in vivo. During the BrdU assay, BrdU is incorporated into replicating DNA and can be detected using anti-BrdU antibodies. After BrdU labeling, an additional DNA hydrolysis step may be required after fixation and permeabilization to allow the anti-BrdU antibody access to the BrdU within the DNA. Besides, BrdU antibodies could be used in conjunction with different kinds of cell type markers.

BrdU Labeling

In Vitro Labeling of Cells with BrdU

1. Prepare a 10 mM stock solution of BrdU by dissolving 3 mg of BrdU in 1 mL water.
2. Dilute the 10 mM BrdU stock solution in cell culture medium to make a 10 µM BrdU labeling solution.
3. Filter the 10 µM BrdU labeling solution through a 0.2 µm filter under sterile conditions.
4. Remove the existing culture medium from the cells and replace with 10 µM labeling solution.
5. Incubate the cells in the BrdU labeling solution for 1-24 h at 37ºC in a CO₂ incubator.

Note: BrdU incubation time depends on how rapidly the cells divide. The exact time required to achieve the optimal signal-to-noise ratio should be optimized.

6. Remove the BrdU labeling solution from the cells. Wash twice in PBS for about 5 seconds per wash.
7. Wash three more times in PBS for two minutes per wash.
8. Fix and permeabilize cells according to standard Immunocytochemistry (ICC)/ Immunofluorescence (IF) protocols.

In Vivo Labeling with BrdU

There are several methods for labeling cells in vivo with BrdU. Two commonly used approaches are oral administration and intraperitoneal injection.

• Oral Administration
1. Dilute BrdU to 0.8 mg/mL in drinking water. Prepare this fresh and change daily.
2. After treatment with BrdU, the animals can then be sacrificed according to standard protocols.
3. Fix and process tissue according to standard Immunohistochemistry (IHC) protocol.

Note: The exact dose should be optimized for individual experimental conditions.

• Intraperitoneal Injection
1. Dilute BrdU in PBS to make a sterile solution of 10 mg/mL.
2. For mice, it is recommended to inject the BrdU solution to a concentration of 100 mg/kg.
3. After treatment with BrdU, the animals can be sacrificed according to standard protocols.
4. Fix and process tissue according to standard IHC protocols.

Note: The exact treatment time and dosage will need to be optimized for your tissue of interest.

DNA Hydrolysis

• Cells
1. Incubate cells in 1-2.5 M HCl for 10 minutes to 1 h at RT.

Note: The exact HCl concentration and incubation time/temperature should be optimized for your experiment.

2. (Optional) Remove the HCl and neutralize with 0.1 M sodium borate buffer (pH 8.5) for 30 minutes at RT.
3. Wash three times in PBS for about 5 seconds per wash.
4. Continue with immunostaining according to standard ICC protocols.
• Tissues Sections
1. Incubate sections in 1-2 M HCl for 30 minutes to 1 h.

Note: The exact HCl concentration and incubation time/temperature should be optimized for your experiment.

2. (Optional) Neutralize sections by incubating in 0.1 M sodium borate buffer (pH 8.5) for 10 minutes at RT.
3. Wash three times in PBS for about 5 seconds per wash.
4. Continue with immunostaining according to standard IHC protocols.

Co-staining with anti-BrdU

BrdU can be used in conjunction with other antibodies to identify proliferating and newly differentiated neurons. See below for our suggestions.

Other Protocols

Antibody Staining of Whole Mount Drosophila Embryos

Primary Cultures for IHC-Viability Assays

Sodium Azide Removal Protocol

Biotin Conjugation