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首页> 外文期刊>Journal of Neuroscience Methods >Vibration enhancement of slide-mounted immunofluorescence staining.
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Vibration enhancement of slide-mounted immunofluorescence staining.

机译：载玻片免疫荧光染色的振动增强。

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Immunofluorescence techniques allow the determination of protein and small molecule distribution within tissues and individual cells. There have been important, innovative modifications of these techniques since their introduction to the biosciences including the use of a mounting medium that prevents photo-bleaching, non-ionic detergents to permeabilize membranes, multiple immunofluorescence labeling and antigen recovery techniques for optimizing ligand-target interactions. While methods have been optimized for ligand-target accessibility in free-floating sections, little innovation has occurred to improve antibody access and epitope recognition in immunohistochemistry on slide-mounted sections or cell culture. During our studies of brain signaling pathways, we sought to improve the immunofluorescence signal to noise ratio in these specimens. We present here a minor modification of immunofluorescence procedures that significantly increases antibody access to epitopes within tissue and improves staining quality while significantly shortening incubation time. Antibody-epitope interactions are dependent on access and affinity. Our technique is based upon application of a vibration source during antibody incubation which increases epitope access, shortens incubation time and thereby minimizes background immunofluorescence. Data are presented on benefits evident with several antibodies raised against proteins and peptides localized in various subcellular compartments. Analysis of the quality of labeling was performed to show that signal intensity is enhanced and background intensity is often diminished when incubations are performed under gentle vibration. This, together with the significant saving of time, should make this procedure applicable to a wide range of neurobiological questions.
机译：免疫荧光技术可以确定组织和单个细胞内蛋白质和小分子的分布。自从将这些技术引入生物科学以来，已经对这些技术进行了重要的创新性修改，包括使用防止光致漂白的固定介质，非离子型去污剂以透化膜，多种免疫荧光标记和抗原回收技术以优化配体-靶标相互作用。尽管针对自由浮动切片中配体-靶标可及性的方法进行了优化，但在载玻片固定切片或细胞培养物的免疫组织化学中，很少有创新来改善抗体的访问和表位识别。在我们研究大脑信号传导途径的过程中，我们试图提高这些样本中的免疫荧光信噪比。我们在这里介绍了免疫荧光程序的一个小修改，可以显着增加抗体对组织内表位的访问，并改善染色质量，同时显着缩短孵育时间。抗体-表位的相互作用取决于进入和亲和力。我们的技术基于在抗体孵育过程中施加振动源，该振动源增加了抗原决定簇的进入，​​缩短了孵育时间，从而使背景免疫荧光最小化。数据显示了几种针对位于不同亚细胞区室的蛋白质和肽产生的抗体的明显益处。标记质量的分析表明，在温和振动下孵育时，信号强度增强并且背景强度通常降低。这与大量的时间节省一起，应使该程序适用于广泛的神经生物学问题。

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