BrdU 标记试剂

可以，EdU和BrdU标记能结合起来在培养细胞和体内用于细胞增殖的双重标记。BrdU将会优先的嵌入到DNA，因此先进行EdU培养再进行BrdU培养。当添加BrdU作为第二标记时，不需要从细胞培养的培养基中移除EdU。进行乙醇固定，随后进行BrdU检测方案中需要的DNA变性的一些方法。之后为EdU检测进行连接标记反应，随后进行用于BrdU检测的抗体标记。确保选择一个不和EdU交叉反应的BrdU抗体，例如我们的MoBU-1克隆（货号B35141）。许多BrdU抗体表现出对EdU一定程度的交叉性反应。此处（https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-proliferation/dual-pulse-labeling-of-cell-proliferation-using-edu-and-brdu-incorporation.html）是使用EdU和BrdU进行双-脉冲标记案例的链接。

Product 00-0103 contains BrdU (5-bromo-2'-deoxyuridine) and FrdU (5-fluoro-2'-deoxyuridine) at a molar ratio of 10:1. The concentrations of BrdU and FrdU in product are approximately 3 mg/mL and 0.3 mg/mL, respectively.

The only study that we performed was in mice injected intraperitoneally with 10 uL of BrdU labeling reagent (catalog #00-0103) per gram of body weight. Incorporation of BrdU was monitored immunohistochemically only in the small intestine. Our data showed that 2-4 hours after BrdU injection in vivo was optimal for detection of staining in small intestine. Positive staining was lighter but still visible 8 hours later, but was gone after 24 hours.

With BrdU injection in mice, it is very unlikely that labeling, at least in small intestine, will last for 3-5 days. It is likely that the BrdU signal intensity as a function of time after labeling is species- and organ-specific, since the proliferation rate of the cells being labeled determines the amount of positive staining at any particular time. Also, labeling efficiency and kinetics are dependent upon the method used to label the cells in the sample with BrdU. For example, BrdU can be administered in vivo by injection, as described above, or by adding it to the animals' drinking water for several days. Labeling kinetics may vary for different types of cells in culture as well. For example, adherent cells typically label with different kinetics than cells that grow in suspension.

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Yes, EdU and BrdU labeling can be combined for dual-pulse labeling of cell proliferation in cultured cells and in vivo. BrdU will be preferentially incorporated into DNA, so perform the EdU incubation first followed by the BrdU incubation. Removal of EdU from the media is not required in cultured cells when BrdU is added as the second label. Perform an alcohol fixation followed by some method of DNA denaturation as required for the BrdU detection protocol and then perform the click labeling reaction for detection of EdU followed by antibody labeling for detection of BrdU. Be sure to select a BrdU antibody that does not have cross-reactivity to EdU, such as our MoBU-1 clone (Cat. No. B35141). Many BrdU antibodies have been shown to have some amount of cross-reactivity with incorporated EdU. Here is a link (http://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-proliferation/dual-pulse-labeling-of-cell-proliferation-using-edu-and-brdu-incorporation.html) to an example protocol for dual-pulse labeling using EdU and BrdU.

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