Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois.
Curr Protoc. 2023 Oct;3(10):e908. doi: 10.1002/cpz1.908.
In single-molecule localization microscopy (SMLM), immunofluorescence (IF) staining affects the quality of the reconstructed superresolution images. However, optimizing IF staining remains challenging because IF staining is a one-step, irreversible process. Sample labeling through reversible binding presents an alternative strategy, but such techniques require significant technological advancements to enhance the dissociation of labels without sacrificing their binding specificity. In this article, we introduce time-lapse imaging of single-antibody labeling. Our versatile technique utilizes commercially available dye-conjugated antibodies. The method controls the antibody concentrations to capture single-antibody labeling of subcellular targets, thereby achieving SMLM through the labeling process. We further demonstrate dual-color single-antibody labeling to enhance the sample labeling density. The new approach allows the evaluation of antibody binding at the single-antibody level and within the cellular environment. This comprehensive guide offers step-by-step instructions for time-lapse imaging of single-antibody labeling experiments and enables the application of the single-antibody labeling technique to a wide range of targets. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for single-antibody labeling Basic Protocol 2: Data acquisition for single-molecule localization microscopy Alternate Protocol: Dual-color single-antibody labeling using OptoSplit II equation Basic Protocol 3: Image analysis.
在单分子定位显微镜（SMLM）中，免疫荧光（IF）染色会影响重构超分辨率图像的质量。然而，优化 IF 染色仍然具有挑战性，因为 IF 染色是一个一步不可逆的过程。通过可逆结合进行样品标记提供了一种替代策略，但此类技术需要重大技术进步来增强标签的解离而不牺牲其结合特异性。本文介绍了单抗体标记的延时成像。我们的多功能技术利用了市售的染料偶联抗体。该方法控制抗体浓度以捕获亚细胞靶标的单抗体标记，从而通过标记过程实现 SMLM。我们进一步展示了双色单抗体标记以增强样品标记密度。新方法允许在单抗体水平和细胞环境中评估抗体结合。本综合指南提供了单抗体标记实验延时成像的分步说明，并使单抗体标记技术能够应用于广泛的目标。© 2023 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案 1：单抗体标记的样品制备基本方案 2：单分子定位显微镜的数据采集备选方案：使用 OptoSplit II 方程的双色单抗体标记基本方案 3：图像分析。
