BrdU staining protocol
Licia Miller Product Manager
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 on cell lines and primary cell cultures in vitro, and on cells in living animals in vivo. During the BrdU assay, BrdU is incorporated into replicated DNA and can be detected using anti-BrdU antibodies.
EdU staining is an alternative method that has a shorter and simpler procedure than BrdU staining.
Stage 1 BrdU labeling
BrdU labeling can be performed both in vitro and in vivo. There are several methods available to label cells with BrdU in vivo, including intraperitoneal injection and oral administration.
• In vitro labeling of cells with BrdU
- BrdU antibody ( such as Ab179916)
-Optional: BrdU control slides as a positive control for BrdU tissue incorporation
Experimental steps
1. Dissolve 3 mg of BrdU in 1 mL of water to prepare a 10 mM BrdU stock solution.
2. Dilute the 10 mM BrdU stock solution with cell culture medium to obtain a 10 µM BrdU labeling solution.
3. Aseptically filter the 10 µM BrdU labeling solution through a 0.2 µm filter.
4. Remove the existing culture medium in the cells and replace with 10 µM labeling solution.
5. Place the cells in BrdU labeling solution and incubate in a CO2 incubator at 37ºC for 1 - 24 hours.
• Tip: The BrdU incubation time depends on how fast the cells divide. Primary cells may require up to 24 hours, while rapidly proliferating cell lines may only require 1 hour. The exact time required to achieve the best signal-to-noise ratio should be optimized.
6. Remove the BrdU labeling solution from the cells and wash twice with PBS, each wash for about 5 seconds.
7. Wash three times with PBS , two minutes each time.
Fix and permeabilize cells according to standard immunocytochemistry (ICC) protocols.
• Before immunostaining, refer to the DNA hydrolysis procedure below.
• In vivo BrdU labeling by intraperitoneal injection
Materials required
- 10 mg/mL BrdU solution in PBS
- BrdU antibody ( such as Ab179916)
-Optional：BrdU control slides as a positive control for BrdU tissue incorporation
Experimental procedures
1. Dilute BrdU in PBS to make a 10 mg/mL sterile solution.
2. For mice, generally, a BrdU solution with a concentration of 100 mg/kg is injected.
• Tip: BrdU can be detected in rapidly dividing tissues (such as the small intestine) as early as 30 minutes after injection. However, for most tissues, you may need to wait up to 24 hours. The exact treatment time and dose will need to be optimized based on your tissue of interest.
3. After treatment with BrdU, animals may be sacrificed according to laboratory-approved procedures.
Fix and process tissues according to standard immunohistochemistry (IHC) protocols.
• Before proceeding with immunostaining, refer to the DNA hydrolysis procedure below.
• In vivo BrdU labeling by oral administration
Oral administration of BrdU is a non-invasive method and can therefore be used to prolong the duration of BrdU administration, although it may introduce variability to the experiment due to the inability to control the animals' water intake.
Materials required
- BrdU stock solution (e.g. 10 mg/mL)
- BrdU antibody ( such as Ab179916)
-Optional: BrdU control slides as a positive control for BrdU tissue incorporation
Experimental steps
1. Dilute BrdU to 0.8 mg/mL in drinking water.
• NOTE: Prepare fresh and replace daily.
• Tip: For mice, 225 mg/kg BrdU per day (calculated by measuring each animal's water intake) should achieve adequate BrdU labeling. However, the exact dose should be optimized based on individual experimental conditions.
2. After treatment with BrdU, animals can be sacrificed according to standard protocols.
Fix and process tissues according to standard IHC protocols.
• However, prior to immunostaining, please refer to the DNA hydrolysis procedure below.
Stage 2 DNA hydrolysis
Following BrdU labeling, an additional DNA hydrolysis step (sometimes called a DNA denaturation step) may be required after fixation and permeabilization to allow the anti-BrdU antibody to access BrdU within the DNA.
Some studies have reported that heat-induced epitope repair may also be performed in place of the HCl hydrolysis step, followed by immunostaining.
• Cell
Materials required
-Sodium Borate Buffer:
- 3.8g sodium borate ( MW=381.4 ) + 100 mL distilled water
- Adjust pH with NaOH
- 1 - 2.5 M hydrochloric acid
Experimental steps
1. Incubate cells in 1 - 2.5 M HCl at room temperature for 10 minutes to 1 hour.
• NOTE: The exact HCl concentration and incubation time should be optimized for your experiments.
If shorter incubation times are used, incubation at 37°C may be more effective than room temperature.
2. Optional step: Remove HCl and neutralize with 0.1 M sodium borate buffer (pH 8.5) at room temperature for 30 minutes.
3. Wash three times with PBS.
Proceed with immunostaining according to standard immunocytochemistry (ICC) protocols.
• Tissue sections
• NOTE: If using paraffin-embedded sections, ensure they are deparaffinized 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 hydrochloric acid
Experimental steps
1. Incubate tissue sections in 1-2 M HCl for 30 minutes to 1 hour.
• NOTE: The exact HCl concentration and incubation time should be optimized for your experiments.
If shorter incubation times are used, incubation at 37°C may be more effective than at room temperature.
2. Optional Step: Neutralizing Tissue Sections.
• Incubate sections in 0.1 M sodium borate buffer (pH8.5) at room temperature for 10 minutes.
3. Wash three times with PBS, each wash for about 5 seconds.
4. Proceed with immunostaining according to standard IHC protocol.
Stage 3 Co-staining with anti-BrdU (optional)
BrdU antibodies can be used together with cell type markers such as Ki67, doublecortin, and NeuN to identify proliferating cells and newly differentiated neurons.
• Ki67
Ki67 protein is a marker of cell proliferation and is present in cells in the G1, S, G2, and M phases of the cell cycle, but not in quiescent (G0) cells. Ki67 antibodies can be used instead of or in combination with BrdU to label proliferating neurons.
• Doublecortin
A microtubule-associated phosphoprotein expressed by immature neurons. Antibodies to doublecortin can be used in conjunction with BrdU to identify immature postmitotic neurons.
• NeuN
A marker for mature neurons that can be used in conjunction with BrdU staining to identify newly differentiated neurons.
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