Quantitative Assessment of Specificity in Immunoelectron Microscopy
Quantitative Assessment of Specificity in Immunoelectron Microscopy
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DOI：
10.1369/jhc.2010.956243
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发表时间：
2010-10-01
影响因子：
3.2
通讯作者：
Gawden-Bone, Christian
中科院分区：
文献类型：
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作者：
Lucocq, John Milton;Gawden-Bone, Christian
在免疫电子显微镜（免疫EM）的切片上，金颗粒用于定位细胞的分子成分。这些颗粒是可计数的，并且已经建立了定量方法来估计和评估来自单个不受控制的标记实验的“原始”金颗粒计数的密度和分布。然而，这些原始计数由两个不同的元素组成：对靶组分具有特异性的颗粒（特异性标记）和不具有特异性的颗粒（非特异性标记）。到目前为止，用于评估特异性标记和校正原始金颗粒计数以揭示特异性标记密度和分布的方法尚未引起太多关注。在这里，我们讨论了确定免疫EM特异性的实验策略，我们提出了定量评估的方法（1）所观察到的金颗粒是特定的目标的概率，（2）特异性标记的密度，（3）在一系列隔室的特异性标记的分布。这些方法应该是一般效用的研究人员调查的细胞成分的分布，使用部分免疫金标记。（J Histochem Cytochem 58：917- 927，2010）
In immunoelectron microscopy (immuno-EM) on ultrathin sections, gold particles are used for localization of molecular components of cells. These particles are countable, and quantitative methods have been established to estimate and evaluate the density and distribution of "raw" gold particle counts from a single uncontrolled labeling experiment. However, these raw counts are composed of two distinct elements: particles that are specific (specific labeling) and particles that are not (nonspecific labeling) for the target component. So far, approaches for assessment of specific labeling and for correction of raw gold particle counts to reveal specific labeling densities and distributions have not attracted much attention. Here, we discuss experimental strategies for determining specificity in immuno-EM, and we present methods for quantitative assessment of (1) the probability that an observed gold particle is specific for the target, (2) the density of specific labeling, and (3) the distribution of specific labeling over a series of compartments. These methods should be of general utility for researchers investigating the distribution of cellular components using on-section immunogold labeling. (J Histochem Cytochem 58:917-927,2010)