BRDU staining

BrdU staining is a widely used method for detecting proliferating cells by incorporating bromodeoxyuridine (BrdU), a synthetic analog of thymidine, into newly synthesized DNA. This protocol from Abcam provides a detailed guide for both in vitro and in vivo BrdU labeling, followed by detection using anti-BrdU antibodies. It supports applications in immunocytochemistry, immunohistochemistry, and ELISA. The method is ideal for researchers studying cell cycle dynamics, tissue regeneration, or cancer proliferation. Abcam offers validated BrdU antibodies and ready-to-use kits, ensuring reproducibility and high sensitivity. Whether you are working with cultured cells or tissue samples, this protocol helps streamline BrdU detection with reliable results. The protocol is also relevant for studies involving humans, though researchers should consider unique limitations such as ethical concerns, detection challenges, and differences in sample processing compared to other model organisms.

Introduction

BrdU (5-bromo-2’-deoxyuridine) is a synthetic nucleoside that integrates into DNA during the S-phase of the cell cycle, making it a powerful tool for identifying proliferating cells. The BrdU staining protocol enables researchers to visualize and quantify DNA synthesis in various biological systems. This protocol outlines step-by-step instructions for BrdU labeling, fixation, DNA denaturation, and antibody-based detection. It is compatible with multiple sample types and experimental designs. This technique is essential in cancer biology, developmental studies, development, adult neurogenesis, and drug screening, offering a robust alternative to traditional proliferation markers. With optimized reagents and kits, Abcam simplifies the BrdU workflow for consistent and accurate results.

Background and principles

The BrdU assay is based on the incorporation of BrdU into replicating DNA during cell division. Once incorporated, BrdU can be detected using specific monoclonal antibodies, allowing researchers to detect and quantify proliferating cells. The protocol involves labeling cells with BrdU, followed by fixation, DNA denaturation (to expose BrdU epitopes), and immunostaining. Detection can be visualized via fluorescence microscopyor quantified using ELISA. Compared to traditional methods such as tritiated thymidine incorporation assays, which use radioactive labeling and scintillation counting to detect DNA synthesis, BrdU offers a non-radioactive and more convenient alternative. This method is highly sensitive and adaptable to both in vitro and in vivo systems.

Stage 1 - BrdU labeling

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

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.
• For rapid, convenient results, we also recommend our BrdU staining IHC kit (ab125306) or BrdU ELISA kit (eg, 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.
• For rapid, convenient results, we also recommend our BrdU staining IHC kit (ab125306) or BrdU ELISA kit (eg 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 a lack of control over an animal’s water consumption.

Materials required

• BrdU stock solution (eg, 10 mg/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.
• For rapid, convenient results, we also recommend our BrdU staining IHC kit (ab125306) or BrdU ELISA kit (eg 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:

  • 3.8 g 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 neuronal nuclear protein expressed by mature post-mitotic neurons. NeuN antibodies can be used to identify differentiated neurons and assess neuronal density.

Comparison to other methods

BrdU staining is often compared to EdU (5-ethynyl-2’-deoxyuridine) staining, another method for detecting DNA synthesis. While EdU offers a faster and simpler protocol using click chemistry, it can also be readily detected without the need for harsh DNA denaturation steps. BrdU remains a gold standard due to its extensive validation and compatibility with archival samples. Unlike Ki-67 or PCNA, which mark general proliferation, BrdU directly measures DNA synthesis, providing more precise temporal resolution. BrdU also allows for dual labeling with other markers.

Experimental design and considerations

Careful experimental design is crucial when using BrdU or EdU assays to study cell proliferation and DNA synthesis. The choice between these thymidine analogs should be guided by the specific goals of your experiment, the characteristics of your cells, and the technical requirements of your detection methods. BrdU, a classic tool for tracking cell cycle progression, is incorporated into newly synthesized DNA during the S phase, making it a reliable marker for identifying proliferating cells. Its extensive validation across a wide range of cell types and experimental conditions makes it a preferred option for many researchers.

However, EdU assays have gained popularity due to their increased sensitivity and streamlined protocols. EdU, like BrdU, is incorporated into DNA during the S phase, but its detection does not require DNA denaturation, preserving cell and tissue morphology and allowing for easier multiplexing with other histological or immunofluorescent markers. This makes EdU particularly suitable for experiments where maintaining specimen integrity is essential or when combining cell proliferation analysis with additional targets.

When planning your experiment, consider factors such as the proliferation rate of your cells, the optimal duration for labeling newly synthesized DNA, and the compatibility of your chosen assay with downstream applications. For rapidly dividing cells, shorter incubation times may suffice, while primary or slowly proliferating cells may require extended labeling periods to ensure robust BrdU or EdU incorporation. Additionally, the sensitivity of your detection method and the need for co-staining with other markers should influence your choice of assay.

Ultimately, selecting the appropriate thymidine analog and optimizing your protocol for your specific cell type and experimental objectives will ensure accurate detection of proliferating cells and reliable measurement of cell cycle dynamics. Thoughtful experimental design not only enhances the quality of your data but also supports reproducibility and comparability across different studies in the field of cellular proliferation.

Applications

BrdU staining is widely used in cell biology, oncology, neuroscience, and developmental biology. It enables researchers to track cell proliferation in cultured cells, tissue sections, and whole organisms. BrdU staining is commonly used to quantify cell number in proliferation assays, providing a reliable measure of cell growth and viability. Stained samples are examined using microscopy or other detection methods to assess marker expression and proliferation. Applications include tumor growth analysis, stem cell tracking, neurogenesis studies, and drug efficacy testing.

Limitations

Despite its utility, BrdU staining has some limitations. The requirement for DNA denaturation can compromise tissue morphology and antigen integrity, potentially affecting co-staining with other markers. BrdU incorporation is also time-sensitive and may vary depending on cell type and proliferation rate. Importantly, BrdU staining does not distinguish between live and dead cells, so additional assays may be required to specifically identify dead cells during cell viability assessments. Furthermore, cell death can affect the interpretation of BrdU staining results, as dying or apoptotic cells may incorporate BrdU differently. Additionally, BrdU is a mutagen and must be handled with care. Compared to EdU, the BrdU protocol is more labor-intensive and time-consuming.

Troubleshooting

Common issues in BrdU staining include weak signal, high background, or poor cell morphology. Weak staining may result from insufficient BrdU incorporation or inadequate DNA denaturation. Ensure optimal BrdU concentration and incubation time based on cell type. High background can stem from non-specific antibody binding; use appropriate blocking buffers and validated antibodies like Abcam’s ab6326. Poor morphology may indicate over-fixation or harsh denaturation, in which case you will need to optimize fixation time and acid treatment.

Including proper controls in BrdU staining experiments is essential for troubleshooting and validating your results. It is also recommended to perform technical replicates to ensure data reliability and reproducibility.

When troubleshooting issues related to staining surface antigens or cell surface markers, especially in protocols involving flow cytometry, ensure that the staining of surface molecules is performed prior to fixation and intracellular staining. If detection issues persist, check the operation of the flow cytometer and review cytometry protocols for accuracy.

We recommend using control slides and kits to validate protocol steps.