Relative labelling index a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections
Relative labelling index a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections
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DOI：
10.1046/j.0022-2720.2001.00977.x
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发表时间：
2002-02-01
影响因子：
2
通讯作者：
Griffiths, G
中科院分区：
文献类型：
--
作者：
Mayhew, TM;Lucocq, JM;Griffiths, G
简单而有效的协议，用于定量免疫金标记的抗原定位在不同的细胞室（细胞器或膜）和统计学评估所产生的标记分布。解决了两个关键问题：（a）实验组（例如对照组或治疗组）内的房室标记是否与随机分布一致？和（B）标记模式在组之间是否不同（例如对照组与处理组）？方案依赖于细胞和隔室的随机取样。躺在指定的细胞器室的金颗粒的数量提供了观察到的频率分布。通过在细胞轮廓上叠加测试点网格，基于设计的体视学用于确定位于这些相同隔室上的点的数量。随机点击中隔室的概率由它们的相对大小决定，因此提供了一个方便的内部标准，即如果标记是纯随机的，则期望分布。通过应用测试线网格，并计算这些交叉膜迹线的位置，类似的程序提供了观察到的和预期的不同类别的膜的标记分布。将观察到的金除以预期的金，得到每个隔室的相对标记指数（RLI），对于随机标记，预测的RLI = 1。与细胞器（golds mum（-2））或膜（golds mum（-1））的标记密度相反，RLI值的估计不需要知道晶格常数（每个点的面积或每个交叉点的长度）或样本放大率。通过卡方分析比较组内的金分布，以测试观察到的分布是否与随机分布显著不同，如果是非随机的，则识别优先标记的区室（RLI > 1）。列联表分析允许比较不同组细胞中的标记分布。协议进行了描述和说明使用工作标本的例子和真实的数据。
Simple and efficient protocols for quantifying immunogold labelling of antigens localized in different cellular compartments (organelles or membranes) and statistically evaluating resulting labelling distributions are presented. Two key questions are addressed: (a) is compartmental labelling within an experimental group (e.g. control or treated) consistent with a random distribution? and (b) do labelling patterns vary between groups (e.g. control vs. treated)? Protocols rely on random sampling of cells and compartments. Numbers of gold particles lying on specified organelle compartments provide an observed frequency distribution. By superimposing test-point lattices on cell profiles, design-based stereology is used to determine numbers of points lying on those same compartments. Random points hit compartments with probabilities determined by their relative sizes and so provide a convenient internal standard, namely, the expected distribution if labelling is purely random. By applying test-line lattices, and counting sites at which these intersect membrane traces, analogous procedures provide observed and expected labelling distributions for different classes of membranes. Dividing observed golds by expected golds provides a relative labelling index (RLI) for each compartment and, for random labelling, the predicted RLI = 1. In contrast to labelling densities of organelles (golds mum(-2)) or membranes (golds mum(-1)), RLI values are estimated without needing to know lattice constants (area per point or length per intersection) or specimen magnification. Gold distributions within a group are compared by chi-squared analysis to test if the observed distribution differs significantly from random and, if it is non-random, to identify compartments which are preferentially labelled (RLI > 1). Contingency table analysis allows labelling distributions in different groups of cells to be compared. Protocols are described and illustrated using worked specimen examples and real data.