Hervás J P, de la Flor J, Santa-Cruz M C
Department of Cell Biology, Faculty of Sciences, Autonomous University of Barcelona, Bellaterra, Spain.
Biotech Histochem. 2002 May;77(3):145-52. doi: 10.1080/bih.77.3.145.152.
The use of bromodeoxyuridine (BrdU) and subsequent immunocytochemical visualization for studying cell proliferation in plant meristems was investigated in Allium cepa L. root-tips. We describe the optimization of an indirect immunoperoxidase method for detecting incorporation of this DNA precursor in pulse-labeled cells. The basic object of this study is to quantify the extent to which the fraction of S-phase cells can reliably be estimated in asynchronous populations. A matrix of parallel labeling schedules with tritiated-thymidine or BrdU was developed, and the labeling indices provided by autoradiography or immunocytochemistry were compared. Thus, 0.5 mM BrdU assured saturation S-phase labeling after an exposure time of 30 min, and the mean length of the S-phase determined under such conditions was similar to that previously reported for this plant system. Interestingly, Feulgen staining did not interfere with subsequent detection of the BrdU probe. This allowed comparative evaluations of the nuclear DNA content by Feulgen-microdensitometry and the position of a given cell in G1, S or G2 compartments. We also explored the possibility of quantifying BrdU-incorporation in single nuclei by densitometry measurement of the peroxidase label.
在洋葱根尖中研究了使用溴脱氧尿苷（BrdU）及随后的免疫细胞化学可视化方法来研究植物分生组织中的细胞增殖。我们描述了一种间接免疫过氧化物酶方法的优化，用于检测这种DNA前体在脉冲标记细胞中的掺入情况。本研究的基本目的是量化在异步群体中可靠估计S期细胞比例的程度。制定了一系列用氚标记胸腺嘧啶核苷或BrdU的平行标记方案，并比较了放射自显影或免疫细胞化学提供的标记指数。因此，0.5 mM BrdU在暴露30分钟后可确保S期标记饱和，在此条件下测定的S期平均长度与该植物系统先前报道的相似。有趣的是，福尔根染色并不干扰随后对BrdU探针的检测。这使得能够通过福尔根显微密度测定法对核DNA含量以及给定细胞在G1、S或G2期的位置进行比较评估。我们还探讨了通过对过氧化物酶标记进行密度测定来量化单个细胞核中BrdU掺入的可能性。
