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首页> 外文期刊>Brain research. Brain research protocols >A brightness-area-product-based protocol for the quantitative assessment of antigen abundance in fluorescent immunohistochemistry.
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A brightness-area-product-based protocol for the quantitative assessment of antigen abundance in fluorescent immunohistochemistry.

机译：基于亮度区域产品的方案，用于荧光免疫组织化学中抗原丰度的定量评估。

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摘要

A problem frequently facing researchers examining abundance of expression of a given antigen is measurement. When the antigen is confined to the nucleus, absolute numbers of nuclei or a percentage of nuclei expressing the antigen in a given region can be estimated. When the antigen is localized to cytoplasm, cytoplasmic organelles or processes or membranes, the assessment becomes more difficult. In these settings, an observer/experimenter may assign a density score but intra- and inter-observer agreement using a three-tiered system, and finer resolution than this, is unlikely to be reproducible. Digital image analysis provides an opportunity to minimize observer bias in quantification of immunohistochemical staining. Previously, reported digital methods have mostly employed chromogen-staining methods and often report mean image brightness. We report a method for quantitatively assessing and expressing abundance of expression of an antigen in neural tissue stained with immunofluorescent methods by determining the brightness-area-product (BAP). The described protocol utilizes simple to use commercially available software and calculates BAP rather than mean brightness as a measure more representative of antigen abundance and visual interpretation. Accordingly, we propose this protocol as a useful adjunct to observer interpretation of fluorescent immunohistochemistry and its application to assessment of antigen abundance for varying patterns of antigen localization.
机译：研究人员经常要检查一个给定抗原的表达量，这是测量的问题。当抗原被限制在细胞核中时，在给定区域中可以估计细胞核的绝对数目或表达抗原的细胞核的百分比。当抗原定位于细胞质，细胞质细胞器或过程或膜时，评估变得更加困难。在这些设置中，观察者/实验者可以分配密度得分，但是使用三层系统的观察者内部和观察者之间的协议以及比这更高的分辨率不太可能重现。数字图像分析提供了将免疫组织化学染色定量中的观察者偏倚最小化的机会。以前，已报道的数字方法主要采用色原染色方法，并且经常报告平均图像亮度。我们报告了一种方法，可以通过确定亮度区域积（BAP）来定量评估和表达免疫荧光方法染色的神经组织中抗原表达的丰度。所描述的协议利用易于使用的市售软件，并计算BAP而不是平均亮度，以更能代表抗原丰度和视觉解释。因此，我们建议该协议作为观察者解释荧光免疫组织化学的有用辅助方法，并将其应用于评估抗原丰度以改变抗原定位方式。

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