Applications of an efficient method for comparing immunogold labelling patterns in the same sets of compartments in different groups of cells
Applications of an efficient method for comparing immunogold labelling patterns in the same sets of compartments in different groups of cells
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DOI：
10.1007/s00418-004-0685-x
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发表时间：
2004-08-01
影响因子：
2.3
通讯作者：
Lucocq, JM
中科院分区：
文献类型：
--
作者：
Mayhew, TM;Griffiths, G;Lucocq, JM
定量免疫电子显微镜通常涉及确定金标记在不同细胞内区室中的分布，然后对同一细胞样本中的区室之间或实验细胞组之间进行比较。在组内比较的情况下，相对标记指数和标记密度估计的最新进展使得测试特定区室是否被优先标记成为可能。这些方法非常适合分析仅限于体积（细胞器）或表面（膜）区室的金标记，但可以进行修改以分析位于两者混合物中的标记。在这里，提出了一种简单有效的方法来对与细胞器和/或膜相关的标签进行组间比较。该方法依赖于对样本进行多级随机采样（通过块和显微镜场），然后简单地计算与不同隔室相关的金颗粒。然后通过列联表分析比较不同组中原金计数的分布，卡方值的统计自由度由隔室的数量和实验细胞组的数量确定。对总卡方值做出重大贡献的区室卡方值可以确定组间主要差异所在。该方法不需要有关隔室大小的信息（例如，细胞器轮廓面积或膜迹线长度），甚至不严格依赖于标准化组间放大倍数。它的应用通过免疫标记研究的数据集进行说明，该研究旨在在电子显微镜水平上定位 KDEL 受体、磷脂酰肌醇 4,5-二磷酸、GLUT4 和 rab4。
Quantitative immunoelectron microscopy often involves determining the distributions of gold label in different intracellular compartments and then drawing comparisons between compartments in the same sample of cells or between experimental groups of cells. In the case of within-group comparisons, recent developments in the estimation of relative labelling index and labelling density make it possible to test whether or not particular compartments are preferentially labelled. These methods are ideally suited to analysing gold label restricted to volume (organelle) or surface (membrane) compartments but may be modified to analyse label localised in mixtures of both. Here, a simple and efficient approach to drawing between-group comparisons for label associated with organelles and/or membranes is presented. The method relies on multistage random sampling of specimens (via blocks and microscopic fields) followed by simply counting gold particles associated with different compartments. The distributions of raw gold counts in different groups are then compared by contingency table analysis with statistical degrees of freedom for chi-squared values being determined by the number of compartments and the number of experimental groups of cells. Compartmental chi-squared values making substantial contributions to the total chi-squared values then identify where the main between-group differences reside. The method requires no information about compartment size (for example, organelle profile area or membrane trace length) and does not even depend critically on standardising between-group magnification. Its application is illustrated using datasets from immunolabelling studies designed to localise the KDEL receptor, phosphatidyl-inositol 4,5-bisphosphate, GLUT4 and rab4 at the electron microscopic level.