BRDU staining

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

• We recommend our BrdU antibody (ab6326), which has been cited in hundreds of publications and independently reviewed by our customers with an average 5-star rating. The clone and its conjugates are manufactured in-house.
• Optional: BrdU control slides as a positive control for tissue incorporation of BrdU (eg ab129956).
• For rapid, convenient results, we also recommend our BrdU staining IHC kit (ab125306) or BrdU ELISA kit (eg ab12556 or ab126572) for a quantitative measure of BrdU incorporation without imaging.

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.

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.

• Before proceeding with immunostaining refer to the DNA hydrolysis step below.

Materials required

• 10 mg/mL BrdU solution in PBS.
• We recommend our BrdU antibody (ab6326), which has been cited in hundreds of publications and independently reviewed by our customers with an average 5-star rating. The clone and its conjugates are manufactured in-house.
• Optional: BrdU control slides as a positive control for tissue incorporation of BrdU (eg ab129956).
• For rapid, convenient results, we also recommend our BrdU staining IHC kit (ab125306) or BrdU ELISA kit (eg ab12556 or ab126572) for a quantitative measure of BrdU incorporation without imaging.

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.

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.

• Before continuing with immunostaining refer to the DNA hydrolysis step below.

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

• BrdU stock solution (eg, 10mg/mL)
• We recommend our BrdU antibody (ab6326), which has been cited in hundreds of publications and independently reviewed by our customers with an average 5-star rating. The clone and its conjugates are manufactured in-house.
• Optional: BrdU control slides as a positive control for tissue incorporation of BrdU (eg ab129956).
• For rapid, convenient results, we also recommend our BrdU staining IHC kit (ab125306) or BrdU ELISA kit (eg ab12556 or ab126572) for a quantitative measure of BrdU incorporation without imaging.

Steps

Dilute BrdU to 0.8 mg/mL in drinking water.

• Prepare this fresh and change daily.

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

Fix and process tissue according to standard IHC protocols.

• However, before immunostaining, refer to the DNA hydrolysis step below.

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

• Sodium borate buffer:

  • 3.8g sodium borate (MW=381.4) + 100 mL distilled water.
  • Adjust pH with NaOH.

• 1–2.5 M HCl

Steps

Incubate cells in 1–2.5 M HCL for 10 minutes to 1 hour 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

• Sodium borate buffer:

  • 3.8 g sodium borate (MW=381.4) + 100 mL distilled water.
  • Adjust pH with NaOH.

• 1–2 M HCl

Steps

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

Optional step: neutralize tissue sections

• Incubate sections in 0.1 M sodium borate buffer pH 8.5 for 10 minutes at room temperature.

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.