解决耳蜗免疫荧光中小鼠对小鼠的问题:用小鼠一抗免疫荧光标记小鼠耳蜗切片的简单双阻断方案

Q1 Agricultural and Biological Sciences Current protocols in mouse biology Pub Date : 2020-10-02 DOI:10.1002/cpmo.84

Mohamed Bassiouni, Katharina Stölzel, Alina Smorodchenko, Heidi Olze, Agnieszka J. Szczepek

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引用次数: 1

摘要

小鼠是听力研究中应用最广泛的动物模型。因此，小鼠耳蜗切片的免疫组织化学和免疫荧光染色仍然是内耳研究的支柱。由于许多一抗在小鼠体内产生，抗小鼠二抗与小鼠自身免疫球蛋白相互作用，产生了“鼠对鼠”的背景问题。在这里，我们描述了小鼠对小鼠背景荧光在出生后小鼠耳蜗切片的模式。此外，我们描述了一种简单的双阻断免疫荧光方案来标记小鼠耳蜗冷冻切片。该方案包含一个常规的血清阻断步骤，以及一个额外的市售抗小鼠IgG阻断试剂阻断步骤。这种阻断技术实际上消除了小鼠耳蜗切片中的“老鼠对老鼠”背景，同时只增加了染色协议的一点时间。我们提供详细的说明和实用提示组织收获，加工和免疫荧光标记。本文描述了进一步的方案修改，以缩短方案的持续时间，基于一抗孵育温度。最后，我们演示了使用市售小鼠单克隆一抗使用不同孵育时间和不同孵育温度进行免疫荧光染色的例子。©2020作者。基本方案:解决耳蜗免疫荧光中小鼠对小鼠的问题:用小鼠抗体对小鼠耳蜗切片进行免疫荧光标记的简单双阻断方案

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Tackling the Mouse-on-Mouse Problem in Cochlear Immunofluorescence: A Simple Double-Blocking Protocol for Immunofluorescent Labeling of Murine Cochlear Sections with Primary Mouse Antibodies

The mouse is the most widely used animal model in hearing research. Immunohistochemistry and immunofluorescent staining of murine cochlear sections have, thus, remained a backbone of inner ear research. Since many primary antibodies are raised in mouse, the problem of “mouse-on-mouse” background arises due to the interaction between the anti-mouse secondary antibody and the native mouse immunoglobulins. Here, we describe the pattern of mouse-on-mouse background fluorescence in sections of the postnatal mouse cochlea. Furthermore, we describe a simple double-blocking immunofluorescence protocol to label mouse cochlear cryosections. The protocol contains a conventional blocking step with serum, and an additional blocking step with a commercially available anti-mouse IgG blocking reagent. This blocking technique virtually eliminates the “mouse-on-mouse” background in murine cochlear sections, while adding only a little time to the staining protocol. We provide detailed instructions and practical tips for tissue harvesting, processing, and immunofluorescence-labeling. Further protocol modifications are described, to shorten the duration of the protocol, based on the primary antibody incubation temperature. Finally, we demonstrate examples of immunofluorescence staining performed using different incubation times and various incubation temperatures with a commercially available mouse monoclonal primary antibody. © 2020 The Authors.

Basic Protocol: Tackling the Mouse-on-Mouse Problem in Cochlear Immunofluorescence: A Simple Double-Blocking Protocol for Immunofluorescent Labeling of Murine Cochlear Sections with Primary Mouse Antibodies

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Current protocols in mouse biology Agricultural and Biological Sciences-Animal Science and Zoology

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet, all too often, nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. The aim of Current Protocols in Mouse Biology is to provide the clearest, most detailed and reliable step-by-step instructions for protocols involving the use of mice in biomedical research. Written by experts in the field and extensively edited to our exacting standards, the protocols include all of the information necessary to complete an experiment in the laboratory—introduction, materials lists with supplier information, detailed step-by-step procedures with helpful annotations, recipes for reagents and solutions, illustrative figures and information-packed tables. Each article also provides invaluable discussions of background information, applications of the methods, important assumptions, key parameters, time considerations, and tips to help avoid common pitfalls and troubleshoot experiments. Furthermore, Current Protocols in Mouse Biology content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Mouse Biology brings together resources in mouse biology and genetics and provides a mouse protocol resource that covers all aspects of mouse biology. Current Protocols in Mouse Biology also permits optimization of mouse model usage, which is significantly impacted by both cost and ethical constraints. Optimal and standardized mouse protocols ultimately reduce experimental variability and reduce the number of animals used in mouse experiments.