전통적으로 세포 주기의 진행 정도는 세포가 S기에 들어갈 때 [3H] thymidine의 양을 측정하여 판단해 왔습니다. [3H] thymidine 정량은 scintillation counting이나 autoradiography에 의해 수행되는데 이 기술은 느릴 뿐만 아니라 방사성 동위원소의 취급 및 폐기가 가능한 사람과 장비가 필요하거나 하는 등의 몇 가지 제한 사항이 있습니다.
이의 대안으로 thymidine과 유사한 화학적 구조를 가지면서도 보다 안정한 bromodeoxyuridine (BrdU)로 [3H] thymidine을 대체할 수 있습니다. BrdU가 활발하게 증식하는 세포의 새로 합성된 DNA 가닥에 통합된 다음에 dsDNA의 변성 과정을 거쳐 면역화학적으로 검출이 가능합니다.
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
1. Prepare a 10 mM stock solution of BrdU (eg ab142567) by dissolving 3 mg of BrdU in 1 mL water.
2. Dilute the 10 mM BrdU stock solution in cell culture medium to make a 10 µM BrdU labeling solution.
3. Filter the 10 µM BrdU labeling solution through a 0.2 µm filter under sterile conditions.
4. Remove the existing culture medium from the cells and replace with 10 µM labeling solution.
5. Incubate the cells in the BrdU labeling solution for 1–24 hours at 37 ºC 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.
6. Remove the BrdU labeling solution from the cells and wash twice in PBS for about 5 seconds per wash.
7. Wash three more times with PBS for two minutes each.
8. Fix and permeabilize cells according to standard immunocytochemistry (ICC) protocols.
- Before proceeding with 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
1. 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.
2. Optional step: remove the HCl and neutralize with 0.1 M sodium borate buffer pH 8.5 for 30 minutes at room temperature.
3. Wash three times in PBS.
4. Continue with immunostaining according to standard immunocytochemistry (ICC) 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.
[출처]
https://www.abcam.com/en-us/technical-resources/protocols/brdu-staining
