BRDU staining

Protocol for identifying proliferating cells using BrdU.

BrdU (bromodeoxyuridine or 5-bromo-2'-deoxyuridine) is an analog of the nucleoside thymidine used in the BrdU assay to identify proliferating cells.

BrdU labeling can be performed  in vitro  for cell lines and primary cell cultures or  in vivo  for labeling cells within a living animal. During the BrdU assay, BrdU is incorporated into replicating DNA and can be detected using anti-BrdU antibodies.

EdU staining is an alternative method with a shorter, simpler protocol than that required for BrdU.

Stage 1 - BrdU labeling

BrdU labeling can be done both  in vitro  and  in vivo. Several methods are available for labeling cells  in vivo with BrdU, including intraperitoneal injection and oral administration.

Materials required

Steps

Prepare a 10 mM stock solution of BrdU (eg ab142567) by dissolving 3 mg of BrdU in 1 mL water.

Dilute the 10 mM BrdU stock solution in a cell culture medium to make a 10 µM BrdU labeling solution.

Filter the 10 µM BrdU labeling solution through a 0.2 µm filter under sterile conditions.

Remove the existing culture medium from the cells and replace with 10 µM labeling solution.

Incubate the cells in the BrdU labeling solution for 1–24 hours at 37ºC in a CO2 incubator.

BrdU incubation time depends on how rapidly the cells divide. Primary cells may need up to 24 hours, while rapidly proliferating cell lines may only need one hour. The exact time required to achieve the optimal signal-to-noise ratio should be optimized.

Remove the BrdU labeling solution from the cells and wash twice in PBS for about 5 seconds per wash.

Wash three more times with PBS for two minutes each.

Fix and permeabilize cells according to standard immunocytochemistry (ICC) protocols.

Materials required

Steps

Dilute BrdU in PBS to make a sterile solution of 10 mg/mL.

For mice, as a general rule, inject the BrdU solution to a concentration of 100 mg/kg.

BrdU incorporation into rapidly dividing tissues, such as the small intestine, will be detectable as soon as 30 minutes post-injection. However, for most tissue, you may need to wait up to 24 hours. The exact treatment time and dosage will need to be optimized for your tissue of interest.

After treatment with BrdU, the animals can be sacrificed according to your lab's approved procedures.

Fix and process tissue according to standard immunohistochemistry (IHC) protocols.

Oral administration of BrdU is a non-invasive procedure and, therefore, useful for extended BrdU administration, although it may introduce variability into experiments due to lack of control over an animal’s water consumption.

Materials required

Steps

Dilute BrdU to 0.8 mg/mL in drinking water.

For mice, 225 mg/kg per day of BrdU (calculated by measuring water consumption volumes per animal) should achieve sufficient BrdU labeling. However, the exact dose should be optimized for individual experimental conditions.

After treatment with BrdU, the animals can then be sacrificed according to standard protocols.

Fix and process tissue according to standard IHC protocols.

Stage 2 - DNA hydrolysis

After BrdU labeling, an additional DNA hydrolysis step (sometimes referred to as a DNA denaturing step) may be required after fixation and permeabilization to allow the anti-BrdU antibody access to the BrdU within the DNA.
Some researchers have reported that they don’t perform the HCl hydrolysis step and simply perform heat-induced epitope retrieval before continuing with immunostaining.

Materials required

Steps

Incubate cells in 1–2.5 M HCL for 10 minutes to 1 hour at room temperature.

The exact HCl concentration and incubation time should be optimized for your experiment.

If using a shorter incubation time, incubating at 37oC may be more effective than at room temperature.

Optional step: remove the HCl and neutralize with 0.1 M sodium borate buffer pH 8.5 for 30 minutes at room temperature.

Wash three times in PBS.

Continue with immunostaining according to standard immunocytochemistry (ICC) protocols.

Note: if using paraffin-embedded sections, ensure they are de-waxed before proceeding.

Materials required

Steps

Incubate tissue sections in 1–2 M HCl for 30 minutes to 1 hour.

The exact HCl concentration and incubation time should be optimized for your experiment.

If using a shorter incubation time, incubating at 37oC may be more effective than at room temperature.

Optional step: neutralize tissue sections

Wash three times in PBS for about 5 seconds per wash.

Continue with immunostaining according to standard IHC protocols.

Stage 3 - Co-staining with anti-BrdU (optional)

BrdU antibodies can be used with cell type markers, such as Ki67, doublecortin, and NeuN, to identify proliferating cells and newly differentiated neurons.

A cellular marker for proliferation, the Ki67 protein is present in cells at cycle phases G1, S, G2, and M but absent in resting (G0) cells.

Ki67 antibodies can be used instead of, or in conjunction with, BrdU to label proliferating neurons.

A microtubule-associated phosphoprotein expressed by immature neurons. Doublecortin antibodies can be used in conjunction with BrdU to identify immature post-mitotic neurons.

A microtubule-associated phosphoprotein expressed by immature neurons. Doublecortin antibodies can be used in conjunction with BrdU to identify immature post-mitotic neurons.