Li Jackson LiangYao, Goh Chi Ching, Ng Lai Guan
Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Singapore, Singapore.
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
Methods Mol Biol. 2018;1763:87-107. doi: 10.1007/978-1-4939-7762-8_9.
The skin is one of the most physiologically important organs where the organism comes into contact with the external environment and is often a site where pathogen entry first occurs. Thus, a better understanding of the specialized cellular behavior of the immune system in the skin may be important for the improved treatment of diseases. Here, we describe in detail a procedure to image the dorsal mouse ear skin, using a customized ear stage and its associated coverslip holder, with an upright multiphoton microscope. As a demonstrative example, we describe the specific protocol for visualizing robust neutrophil trafficking in albino lysozyme-EGFP mice in response to zymosan particles. Instructive sections are provided for the mouse ear preparation, intradermal delivery of zymosan, design and use of the custom ear stage, as well as a solution for the uninterrupted live imaging of mice during prolonged sessions within a dark box. The mouse ear is easily accessible for imaging, and unlike most other organs, does not require any invasive surgery to be performed.
皮肤是机体与外部环境接触的最重要的生理器官之一，且常常是病原体首先侵入的部位。因此，更好地了解皮肤中免疫系统的特殊细胞行为对于改善疾病治疗可能很重要。在这里，我们详细描述了一种使用定制的耳部载物台及其相关盖玻片固定器，通过直立式多光子显微镜对小鼠背部耳部皮肤进行成像的方法。作为一个示范例子，我们描述了在白化病溶菌酶 - 增强绿色荧光蛋白（EGFP）小鼠中可视化对酵母聚糖颗粒产生的强大中性粒细胞迁移的具体方案。文中提供了关于小鼠耳部制备、酵母聚糖皮内注射、定制耳部载物台的设计与使用的指导性章节，以及在暗箱中长时间对小鼠进行不间断实时成像的解决方案。小鼠耳部易于成像，并且与大多数其他器官不同，不需要进行任何侵入性手术。
