While conventional antibodies have been an instrument of choice in immunocytochemistry for some time, their small counterparts known as nanobodies have been much less frequently used for this purpose. In this study we took advantage of the availability of nanobody cDNAs to site-specifically introduce a non-standard amino acid carrying an azide/alkyne moiety, allowing subsequent Cu(I)-catalyzed Azide-Alkyne Click Chemistry (CuAAC). This generated a fluorescently labelled nanobody that can be used in single step immunocytochemistry as compared to conventional two step immunocytochemistry. Two strategies were explored to label nanobodies with Alexa Fluor 488. The first involved enzymatic addition of an alkyne-containing peptide to nanobodies using sortase A, while the second consisted of incorporating para-azido phenylalanine at the nanobody C-terminus. Through these approaches, the fluorophore was covalently and site-specifically attached. It was demonstrated that cortactin and β-catenin, cytoskeletal and adherens junction proteins respectively, can be imaged in cells in this manner through single step immunocytochemistry. However, fixation and permeabilization of cells can alter native protein structure and form a dense cross-linked protein network, encumbering antibody binding. It was shown that photoporation prior to fixation not only allowed delivery of nanobodies into living cells, but also facilitated β-catenin nanobody Nb86 imaging of its target, which was not possible in fixed cells. Pharmacological inhibitors are lacking for many non-enzymatic proteins, and it is therefore expected that new biological information will be obtained through photoporation of fluorescent nanobodies, which allows the study of short term effects, independent of gene-dependent (intrabody) expression.

中文翻译：

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纳米抗体点击化学用于方便的位点特异性荧光标记、单步免疫细胞化学和通过光穿孔和活细胞成像递送到活细胞中

虽然一段时间以来，常规抗体一直是免疫细胞化学的首选工具，但它们被称为纳米抗体的小型对应物很少用于此目的。在这项研究中，我们利用纳米抗体 cDNA 的可用性来定点引入带有叠氮化物/炔烃部分的非标准氨基酸，从而允许随后的 Cu(I) 催化的叠氮化物-炔烃点击化学 (CuAAC)。与传统的两步免疫细胞化学相比，这产生了一种荧光标记的纳米抗体，可用于单步免疫细胞化学。探索了两种策略来使用 Alexa Fluor 488 标记纳米抗体。 第一种涉及使用分选酶 A 将含炔肽的酶促添加到纳米抗体中，而第二种包括在纳米抗体 C 端加入对叠氮基苯丙氨酸。通过这些方法，荧光团被共价地和位点特异性地附着。结果表明，通过单步免疫细胞化学，可以以这种方式在细胞中分别对 cortactin 和 β-catenin、细胞骨架和粘附连接蛋白进行成像。然而，细胞的固定和透化可以改变天然蛋白质结构并形成密集的交联蛋白质网络，阻碍抗体结合。结果表明，固定前的光穿孔不仅允许将纳米抗体递送到活细胞中，而且还促进了其靶标的 β-连环蛋白纳米抗体 Nb86 成像，这在固定细胞中是不可能的。许多非酶蛋白缺乏药理学抑制剂，