Kramer Bernhard A, Del Castillo Jacobo Sarabia, Pelkmans Lucas, Gut Gabriele
Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.
Molecular Life Sciences PhD Program, Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland.
Bio Protoc. 2023 Jul 5;13(13):e4712. doi: 10.21769/BioProtoc.4712.
Highly multiplexed protein measurements from multiple spatial scales using fluorescence microscopy recently emerged as a powerful way to investigate tumor microenvironments in biomedicine and the multivariate nature of complex systems' interactions. A range of methods for this exist, which either rely on directly labeling the primary antibody with oligonucleotides/rare metals or employing methods to remove fluorescence for cyclic acquisition. Here, we describe a protocol that uses off-the-shelf primary and secondary antibodies without further need for modification and only commonly available chemical reagents. The method harnesses the observation that antibodies can crosslink to bound epitopes during light exposure, thus preventing elution. By utilizing a simple oxygen radical scavenging buffer during imaging and by blocking free sulfhydryl groups before antibody incubation, the presented method can employ comparably mild conditions to remove bound antibodies from epitopes, which preserves sample integrity. Thus, with the stated minor modifications, it allows for a standard immunofluorescence imaging protocol in cyclic fashion, currently permitting staining of up to ~80 unique epitopes.
近年来，利用荧光显微镜在多个空间尺度上进行高度多重的蛋白质测量，已成为生物医学中研究肿瘤微环境以及复杂系统相互作用的多变量性质的一种强大方法。目前存在一系列用于此目的的方法，这些方法要么依赖于用寡核苷酸/稀有金属直接标记一抗，要么采用去除荧光以进行循环采集的方法。在此，我们描述了一种方案，该方案使用现成的一抗和二抗，无需进一步修饰，仅使用常见的化学试剂。该方法利用了这样一个观察结果：抗体在光照期间可与结合的表位交联，从而防止洗脱。通过在成像过程中使用简单的氧自由基清除缓冲液，并在抗体孵育前封闭游离巯基，本方法可采用相对温和的条件从表位上去除结合的抗体，从而保持样品完整性。因此，通过上述微小修改，它允许以循环方式进行标准免疫荧光成像方案，目前可对多达约80个独特表位进行染色。
