Low doses of bromo- and iododeoxyuridine produce near-saturation labeling of adult proliferative populations in the dentate gyrus
Low doses of bromo- and iododeoxyuridine produce near-saturation labeling of adult proliferative populations in the dentate gyrus
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DOI：
10.1111/j.1460-9568.2005.03907.x
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发表时间：
2005-02-01
影响因子：
3.4
通讯作者：
Kuan, CY
中科院分区：
文献类型：
--
作者：
Burns, KA;Kuan, CY
细胞增殖可以通过在祖细胞中DNA合成期间掺入氚化胸苷（3 H-dT）或卤代嘧啶来检测。在不同时间给药两种胸苷类似物可以进一步确定增殖细胞的细胞周期动力学。传统上，这是通过结合溴脱氧尿苷（BrdU）免疫细胞化学和3 H-dT放射自显影，或通过BrdU和碘脱氧尿苷（IdU）双标记使用两种小鼠抗体。然而，这些方法要么需要漫长的暴露时间，要么涉及用于区分相同物种的两种抗体的复杂的组织学程序。在这里，我们报告了一个简单而可靠的方法区分BrdU和IdU标记的细胞免疫荧光。该方法使用识别BrdU和IdU的小鼠单克隆抗体和与IdU无交叉反应性的大鼠抗BrdU抗体。当结合与不同的荧光团共轭的物种特异性的二抗，这种方法确定BrdU-和IdU-掺入作为双重和单一标记的细胞，分别。该方法具有广泛的应用。首先，我们证明了这种方法可以区分不同胚胎日产生的小鼠皮层神经元。其次，通过以不同的时间间隔给予IdU和BrdU，我们使用这种方法计算出成年小鼠齿状回中神经祖细胞的S期长度约为6 h。最后，我们表明，六倍高浓度的IdU检测到的细胞比标准剂量的BrdU（50毫克/公斤），使用双标记方法只有10%。这些结果表明，标准剂量的BrdU是足够的，以标记大多数的增殖群体在S期脉冲标记实验。
Cell proliferation can be detected by the incorporation of tritiated thymidine (3H-dT) or halopyrimidines during DNA synthesis in progenitor cells. Administration of two thymidine analogues at different times can further determine the cell-cycle kinetics of proliferating cells. Traditionally, this was done by combining bromodeoxyuridine (BrdU) immunocytochemistry and 3H-dT autoradiography, or by BrdU and iododeoxyuridine (IdU) double-labeling using two mouse antibodies. However, these methods either require lengthy exposure time or involve complicated histological procedures for differentiating between two antibodies of the same species. Here we report a simple and reliable method of distinguishing BrdU- and IdU-labeled cells by immunofluorescence. This method uses a mouse monoclonal antibody that recognizes both BrdU and IdU and a rat anti-BrdU antibody that has no cross-reactivity with IdU. When combined with species-specific secondary antibodies that are conjugated to different fluorophores, this method identifies BrdU- and IdU-incorporation as doubly and singly labeled cells, respectively. This method has broad applications. First, we demonstrate that this method can distinguish mouse cortical neurons generated on different embryonic days. Second, by administering IdU and BrdU at varying intervals, we used this method to calculate that the length of S-phase of neural progenitor cells in the adult mouse dentate gyrus is approximately 6 h. Finally, we show that a six-fold higher concentration of IdU detects only 10% more cells than the standard dose of BrdU (50 mg/kg) using the double-labeling method. These results suggest that the standard dose of BrdU is sufficient to label the majority of proliferative populations in the S-phase in pulse labeling experiments.