Bright-field microscopy visualization of proteins and protein complexes by in situ proximity ligation with peroxidase detection.
Bright-field microscopy visualization of proteins and protein complexes by in situ proximity ligation with peroxidase detection.
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DOI：
10.1373/clinchem.2009.134452
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发表时间：
2010
影响因子：
9.3
通讯作者：
Agata Zięba;Carolina Wählby;F. Hjelm;L. Jordan;J. Berg;U. Landegren;Katerina Pardali
中科院分区：
文献类型：
--
作者：
Agata Zięba;Carolina Wählby;F. Hjelm;L. Jordan;J. Berg;U. Landegren;Katerina Pardali
原位邻位连接测定（PLA）允许将蛋白质或蛋白质复合物表示为可扩增的DNA分子。识别是由与寡核苷酸偶联的抗体组成的邻近探针介导的。在邻近探针的双重结合后，寡核苷酸指导环状DNA分子的形成，然后通过滚环复制扩增。然后用荧光探针检测定位的多联体产物。原位PLA能够在固定的细胞或组织切片中定位检测单个天然蛋白质或相互作用的蛋白质对，从而为基础和临床研究提供重要工具。方法：我们使用辣根过氧化物酶（HRP）标记的寡核苷酸偶联原位PLA与酶可视化的本地化检测事件。结果我们证明了蛋白质复合物的检测，无论是在细胞和组织切片，并表明，我们可以量化的复合物与图像分析软件专门开发的识别HRP信号在明场显微镜图像。我们表明，荧光和HRP信号产生相同的结果，无论是在培养的细胞和组织样本。结论：原位PLA与明场检测和自动图像分析的结合允许以自动方式对存在的信号进行计数，从而提供了一种灵敏和特异的方法，用于用明场显微镜定量蛋白质和蛋白质复合物。通过这种方法，可以使用原位PLA而不需要昂贵的荧光显微镜，从而避免了非特异性荧光的问题，同时保持与传统组织学染色的兼容性。
BACKGROUND The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes, the oligonucleotides direct the formation of a circular DNA molecule, which is then amplified by rolling-circle replication. The localized concatemeric product is then detected with fluorescent probes. The in situ PLA enables localized detection of individual native proteins or interacting protein pairs in fixed cells or tissue sections, thus providing an important tool for basic and clinical research. METHODS We used horseradish peroxidase (HRP)-conjugated oligonucleotides to couple in situ PLA with enzymatic visualization of the localized detection event. RESULTS We demonstrate the detection of protein complexes, both in cells and in tissue sections, and show that we can quantify the complexes with image-analysis software specially developed for recognizing HRP signals in bright-field microscopy images. We show that fluorescence and HRP signals produce equivalent results, both in cultured cells and in tissue samples. CONCLUSIONS The combination of in situ PLA with bright-field detection and automated image analysis allows the signals present to be counted in an automated fashion and thus provides a sensitive and specific method for quantification of proteins and protein complexes with bright-field microscopy. With this approach, in situ PLA can be used without the requirement for expensive fluorescence microscopes, thereby avoiding problems with nonspecific fluorescence while maintaining compatibility with conventional histologic staining.