McGowan Jeremy W D, Bidwell Gene L
Department of Neurology, The University of Mississippi Medical Center.
Department of Neurology, The University of Mississippi Medical Center;
J Vis Exp. 2016 Dec 24(118):54987. doi: 10.3791/54987.
Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions. Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues. Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system.
荧光标记是一种成熟的方法，用于在体外和体内的各种实验条件下检查标记分子的命运。荧光探针在确定所给药大分子的生物分布方面特别有用，因为添加小分子荧光标记不太可能影响化合物的动力学或生物分布。存在多种检查生物分布的方法，这些方法在所需工作量以及所得测量是否完全定量方面有很大差异，但结合使用多种方法可以提供一个快速有效的生物分布分析系统。离体全器官成像方法可用于快速比较组织内以及多种组织类型或治疗组之间荧光分子的相对浓度。使用专为活体动物或全器官成像设计的成像平台，可以在不进行进一步处理的情况下确定完整组织内的荧光，节省时间和劳动力，同时提供整体生物分布的准确图像。此过程在试图确定化合物的组织特异性或比较多种不同化合物的实验中非常理想。另一方面，定量组织组织学需要对组织进行广泛的进一步处理，以便对标记化合物进行定量测量。为了准确评估生物分布，必须将所有感兴趣的组织切片、扫描并相对于标准曲线进行分析，以便在组织或组之间进行比较。定量组织组织学是确定组织内绝对化合物浓度的金标准。在此，我们描述了如何有效地将这两种方法结合使用，以评估不同给药方法和化合物修饰将荧光标记分子靶向并递送至中枢神经系统的能力。
