Click Chemistry for Visualization of Newly Synthesized RNA and Antibody Labeling on Ultrathin Tissue Sections.
Click Chemistry for Visualization of Newly Synthesized RNA and Antibody Labeling on Ultrathin Tissue Sections.
复制标题
单击 Chemistry 可在超薄组织切片上可视化新合成的 RNA 和抗体标记。
DOI：
10.1093/micmic/ozad067.552
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Ostroff,Linnaea
中科院分区：
文献类型：
--
作者：
Pérez-Garza,Janeth;Orea,Jairo;Ostroff,Linnaea
点击化学包括快速、选择性、高产率、无副反应或副产物的合成反应[1]。由于点击反应是双正交的，在温和的条件下发生，它们与生物系统兼容，并可与核酸[2]和蛋白质[3]的代谢标记相结合。对于信使核糖核酸的代谢标记，活细胞或组织用尿苷类似物处理，该尿苷类似物含有可用于捕获标记信使核糖核酸或显示其在原位分布的官能团[4]。官能团的检测通常是用抗体进行的，但点击化学为显微镜提供了更好的灵敏度、特异性和空间分辨率[2]。信使核糖核酸的代谢标记对研究脑组织的可塑性变化特别感兴趣，这涉及到复杂的基因表达级联和亚细胞信使核糖核酸定位的精确时空调节[5]。关于活的大脑中转录和翻译的动力学，特别是关于神经元过程中信使核糖核酸的运输动力学，仍有许多悬而未决的问题[5，6]。基于点击化学的信使核糖核酸可以用5-乙炔基尿苷(5-Eu)进行标记，5-Eu是一种炔基功能化尿苷。然后，通过铜(I)催化的炔-叠氮环加成(CuAAC)反应，将其与叠氮共轭标签(如荧光团或生物素)反应，即可显示含Eu的mRNA[2，4]。这种方法已经被用来在小鼠[7]和斑马鱼[8]的荧光显微镜下显示脑组织中Eu标记的mRNA，但脑组织的密度排除了神经元突起中标记的mRNA的荧光成像，这只能在体外分离的神经元中完成[8]。包埋前的电子显微镜(EM)[9]已经在活体轴突中检测到了Eu标记的mRNA，但这种方法非定量，对大面积检测效率低，并且难以与免疫标记等其他检测方法相结合。与标准厚度的切片相比，树脂包埋组织的超薄切片为荧光显微镜提供了更高的空间分辨率，同时在成像规模和多路标记方面提供了更大的灵活性。为了优化在脊椎动物大脑超薄切片上显示Eu的方法，我们通过插管将Eu单次立体定向注入成年大鼠的杏仁外侧核。30min后，经心脏灌流乙醛固定剂固定大脑，并将含有杏仁核的振动刀切片嵌入甲基丙烯酸酯树脂中。树脂中不包含交联剂，因此在标记之前，切片可以很容易地去塑化。在小鼠脑中检测到Eu的两篇文章依赖于商业试剂盒，这些试剂盒价格昂贵，故障排除困难。为了避免使用试剂盒，我们采用了一种在细菌[10]中标记Eu的方法，该方法在新鲜固定的(图1a)和超薄的树脂包埋切片(图1b)中都检测到了Eu。通过超薄切片上的点击化学和免疫荧光，我们能够将新合成的mRNA与树突标记MAP2(图1C)和轴突标记SMI312(图1D)共定位。除了代谢标记，点击化学在免疫组织化学的信号放大方面具有潜在的优势。为了增加检测每个初级抗体分子的探针分子(如荧光团)的数量，信号放大方法的策略要么依赖于多层标记的亲和探针，如链霉亲和素和生物素，要么依赖于酶催化的报告沉积。这两种策略都会降低空间分辨率，前者是因为…
Click chemistry consists of synthesis reactions that are rapid, selective, high-yield, and have no side reactions or byproducts [1]. Because click reactions are biorthogonal and occur under mild conditions, they are compatible with biological systems and can be combined with metabolic labeling of nucleic acids [2] and proteins [3]. For metabolic labeling of mRNA, living cells or tissue are treated with a uridine analog bearing a functional group that can be used to capture labeled mRNA or to visualize its distribution in situ [4]. Detection of the functional group is typically performed with an antibody, but click chemistry provides better sensitivity, specificity, and spatial resolution for microscopy [2]. Metabolic labeling of mRNA is of particular interest in studying plastic changes in brain tissue, which involve complex gene expression cascades and precise spatiotemporal regulation of subcellular mRNA localization [5]. Many open questions remain about the dynamics of transcription and translation in the living brain, especially with respect to the kinetics of mRNA trafficking within neuronal processes [5, 6]. Click chemistry-based mRNA labeling can be performed with 5-ethynyl uridine (5-EU), an alkyne-functionalized uridine. EU-containing mRNA can then be visualized by reacting it with an azide-conjugated tag, such as a fluorophore or biotin, via a copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction [2, 4]. This approach has been used to visualize EU-labeled mRNA in brain tissue by fluorescence microscopy in mice [7] and zebrafish [8], but the density of brain tissue precludes fluorescence imaging of labeled mRNA in neuronal processes, which could only be done in isolated neurons in vitro [8]. EU-labeled mRNA has been detected in axons in vivo by pre-embedding electron microscopy (EM)[9], but this approach is non-quantitative, inefficient for examining large areas, and is difficult to combine with other detection methods such as immunolabeling. Ultrathin sections of resin-embedded tissue provide greatly enhanced spatial resolution for fluorescence microscopy relative to sections of standard thickness while offering more flexibility in imaging scale and multiplexed labeling. To optimize a protocol for visualizing EU on ultrathin sections of vertebrate brain, we performed a single stereotactic infusion of EU into the lateral amygdala of adult rats via a cannula. After allowing 30 min for the EU to be incorporated, the brains were fixed by transcardial perfusion of aldehyde fixatives and vibratome sections containing the amygdala were embedded in methacrylate resin. No crosslinker was included in the resin so that sections could be easily de-plasticized before labeling. The two publications that detected EU in mouse brain relied on commercial kits, which are expensive and difficult to troubleshoot. To avoid the use of kits, we adapted a protocol used to label EU in bacteria [10], which detected EU in both fresh fixed (Figure 1a) and ultrathin resin embedded sections (Figure 1b). By following click chemistry with immunofluorescence on ultrathin sections, we were able to colocalize newly synthesized mRNA with the dendritic marker MAP2 (Figure 1c) and the axonal marker SMI312 (Figure 1d). In addition to metabolic labeling, click chemistry potentially has an advantage in signal amplification for immunohistochemistry. To increase the number of probe molecules (eg, fluorophores) that detect each primary antibody molecule, signal amplification methods strategies rely either on multiple layers of labeled affinity probes, such as streptavidin and biotin, or on enzyme-catalyzed reporter deposition. Both strategies degrade spatial resolution, the former because of the …
影响因子：
14.8
作者：
Alvarez-Castelao, Beatriz;Schanzenbaecher, Christoph T.;Schuman, Erin M.
通讯作者：
Schuman, Erin M.
影响因子：
5.7
作者：
Das, Sulagna;Singer, Robert H.;Yoon, Young J.
通讯作者：
Yoon, Young J.