Department of Neurology, Washington University School of Medicine, St. Louis, Missouri.
Curr Protoc. 2021 Aug;1(8):e214. doi: 10.1002/cpz1.214.
Immunofluorescence labeling and microscopy offer a highly specific means to visualize proteins or other molecular species in a sample by labeling target antigens with fluorescent probes. These fluorescent probes can then be visualized using a fluorescence microscope, allowing their relative spatial relationships to be determined. Due to spectral overlap of common fluorophores, however, it can be challenging to analyze more than three antigens in a single sample with standard imaging approaches. This article describes multiplexed labeling and imaging of four target antigens through the use of a long-Stokes-shift fluorophore-a fluorophore with an unusually large gap between its excitation and emission maxima-in tandem with three conventional fluorophores. This combination allows for multiplexed imaging of four antigens in a single sample with excellent spectral discrimination suitable for sensitive analyses using standard imaging hardware. Particular advantages of this approach are its flexibility in terms of target antigens and the lack of any specialized procedures, reagents, or equipment beyond the commercially available labeling reagent coupled to the long-Stokes-shift fluorophore. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Four-probe immunofluorescence labeling Basic Protocol 2: Four-probe immunofluorescence imaging.
免疫荧光标记和显微镜技术通过用荧光探针标记靶抗原，提供了一种在样本中可视化蛋白质或其他分子种类的高度特异性方法。然后可以使用荧光显微镜观察这些荧光探针，从而确定它们的相对空间关系。然而，由于常见荧光团的光谱重叠，使用标准成像方法在单个样本中分析超过三个抗原可能具有挑战性。本文通过使用长斯托克斯位移荧光团（一种激发和发射最大值之间存在异常大间隙的荧光团）与三种常规荧光团结合，描述了对四个靶抗原进行的多重标记和成像。这种组合允许在单个样本中对四个抗原进行多重成像，具有出色的光谱分辨率，适合使用标准成像硬件进行灵敏分析。该方法的特别优点是其在靶抗原方面的灵活性，并且除了与长斯托克斯位移荧光团偶联的市售标记试剂之外，不需要任何特殊的程序、试剂或设备。
