A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry
A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry
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DOI：
10.1016/s1385-299x(02)00201-5
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发表时间：
2002-10-01
期刊：
影响因子：
--
通讯作者：
Sundstrom, L
中科院分区：
文献类型：
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作者：
Brana, C;Benham, C;Sundstrom, L
荧光排斥染料碘化丙啶（PI）被广泛用作组织培养系统中的活体染料，并标记缺乏完整质膜的垂死细胞中的细胞核。我们已经开发了一种方法，它允许在多聚甲醛固定的组织中保存PI信号，从而能够随后对标记细胞进行免疫组织化学表征。我们已经测试了这种方法在缺血模型的基础上剥夺氧和葡萄糖的器官型海马切片文化，结合免疫细胞化学检测钙蛋白酶-I介导的血影蛋白分解产物（BDPs）。使用共聚焦激光显微镜，它是可能的，在单细胞水平的PI阳性细胞和细胞表达的BDP。该方法也可与其他免疫细胞化学标记物一起使用，以确定在体外积累PI的细胞的表型。通过在损伤后的不同时间固定组织，可以在实验方案期间的任何时间获得活力的“快照”，并随后将在固定时积累PI的那些细胞固定。该技术也可以证明在其他体外和体内系统中表征细胞死亡是有用的。(C)2002 Elsevier Science B. V.保留所有权利。
The fluorescent exclusion dye propidium iodide (PI) is widely used as a vital dye in tissue culture systems and labels the nucleus in dying cells which lack an intact plasma membrane. We have developed a method, which allows the preservation of the PI signal in paraformaldehyde-fixed tissue, enabling subsequent immunohistochemical characterisation of labelled cells. We have tested this method in a model of ischemia based on oxygen and glucose deprivation in organotypic hippocampal slice cultures, in combination with immunocytochemical detection of calpain-I mediated spectrin breakdown products (BDPs). Using confocal laser microscopy it was possible to correlate at the single cell level which cells were PI positive and which cells expressed BDPs. This method can also be used with other immunocytochemical markers to determine the phenotype of cells, which accumulate PI in vitro. By fixing tissue at different times after insults, it is possible to obtain a 'snapshot' of viability at any time during the experimental protocol and subsequently characterise those cells which had accumulated PI at the time of fixation. The technique may also prove useful in characterising cell death in other in vitro and in vivo systems. (C) 2002 Elsevier Science B.V. All rights reserved.