Figure 1: BrdU

Introduction to BrdU

BrdU Function

• BrdU is a thymidine analog that is incorporated into cells during cell proliferation.
• Immunocytochemical detection of bromodeoxyuridine (BrdU) incorporated into DNA is a powerful tool for studying the dynamics of normal and tumor cell proliferation.
• Labeling tumor cells with BrdU, either in vitro or in vivo, followed by detection with specific anti-BrdU monoclonal antibodies, is an accurate and comprehensive method for quantifying the degree of DNA synthesis.

BrdU Localization

• Cell nucleus

Figure 2: Results of the ICC experiment with BrdU, using the Anti-BrdU [BU1/75 (ICR1)] antibody product (ab6326). Green: BrdU, Red: alpha tubulin, Blue: DAPI.

IHC experiment tips

Precautions

• When using intraperitoneal injection of BrdU for in vivo labeling, for rapidly dividing tissues (such as the small intestine), BrdU incorporation is fast and can reach detection levels within 30 minutes after injection; for most tissues, labeling may require a waiting time of up to 24 hours. The exact processing time and dosage need to be optimized according to the tissue type.
• When using oral BrdU for in vivo labeling, for example in mice, a daily dose of 225 mg/kg of BrdU (calculated by measuring the water consumption of each animal) should be sufficient for labeling. The exact dosage needs to be optimized for specific experimental conditions.
• BrdU antibody recognizes single-stranded DNA, so the antibody needs to first denature the double-stranded DNA. DNAse can be used to denature the double-stranded DNA, and 2 M hydrochloric acid denaturation or heat denaturation is the most effective depending on the assay method. Please note that this step is crucial in any experiment using these antibodies.
• The above and more detailed BrdU usage methods can be obtained through the following website:
  https://www.abcam.cn/protocols/brdu-staining-protocol
• Some BrdU antibodies may cross-react with CldU, IdU, or EdU. Please refer to the product manual for specific information.

Positive control

• Rat small intestine tissue treated with BrdU

Example results

Figure 3: IHC- Anti-BrdU [BU1/75 (ICR1)] antibody product (ab6326).

Sample name: Rat small intestine paraffin section.
Primary antibody: Anti-BrdU [BU1/75 (ICR1)] (ab6326) primary antibody, concentration 3 µg/ml.
Antigen retrieval method: Heat in a pressure cooker, antigen retrieval with sodium citrate buffer (pH 6) for 30 minutes.

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample fixation:

1. The fixation time of the sample depends on the size of the tissue block and the type of tissue, but for most samples, room temperature fixation for 18-24 hours is more appropriate.
2. Excessive fixation will close antigenic epitopes. Although antigen retrieval will expose some of the epitopes, if the tissue fixation time is very long (such as more than a week), there will still be no signal after antigen retrieval.
3. For IHC-Fr experiments using perfusion-fixed tissues, no further fixation is required after sectioning; if fresh tissues frozen with isopentane are used, the sections need to be fixed with 4°C pre-cooled PFA or methanol for 10 minutes after sectioning.

Antigen retrieval:

1. When performing immunohistochemistry experiments on paraffin sections, we recommend using a pressure cooker for heat-induced antigen retrieval. For example, 110°C for 15 minutes.
2. When performing immunohistochemistry experiments on frozen sections, if the samples have been fixed with aldehydes for 18-24 hours in advance, enzyme antigen retrieval can be attempted, but please optimize the enzyme concentration and retrieval time to avoid damaging the tissue morphology of the sections.

ICC experiment tips

Precautions

• The labeling time of BrdU depends on the speed of cell division. For primary cells, labeling may take up to 24 hours, while for rapidly proliferating cell lines, labeling may only take one hour. To achieve optimal results, the labeling time of BrdU needs to be optimized according to the cell type.
• BrdU antibody recognizes single-stranded DNA, so the antibody incubation needs to first denature the double-stranded DNA. Denaturation with 2 M HCl or heat denaturation is the most effective, depending on the assay method. Please note that this step is crucial in any experiment using these antibodies.
• The above and more detailed BrdU usage methods can be found by clicking on the following link:
  https://www.abcam.cn/protocols/brdu-staining-protocol
• Some BrdU antibodies may cross-react with CldU, IdU, or EdU. Please refer to the product manual for specific information.
• It is recommended to use 0.1% Triton-X to permeabilize cells.

Positive control

• HeLa cells treated with BrdU.

Example of results

Figure 4: ICC-Anti-BrdU [IIB5] antibody product (ab8955).

Sample name: BrdU-labeled MR65 lung cancer cells.
Primary antibody: Anti-BrdU [IIB5] primary antibody, diluted at a concentration of 1:1000.

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample fixation:

1. Fixation with 4% PFA is recommended, and it is recommended to fix at room temperature for 10-20 minutes.

Sample permeabilization:

1. We recommend using 0.1-0.25% Triton-X 100 for permeabilization at room temperature for 10 minutes.

Antibody incubation:

1. We recommend optimizing the antibody dilution according to the antibody instructions in the early stage of the experiment.

References

1. Frade JM, Ovejero-Benito MC. Neuronal cell cycle: the neuron itself and its circumstances. Cell Cycle. 2015;14(5):712-20. doi: 10.1080/15384101.2015.1004937.
2. Wojcik A, Bruckmann E, Obe G. Insights into the mechanisms of sister chromatid exchange formation. Cytogenet Genome Res. 2004;104(1-4):304-9. doi: 10.1159/000077507.