Department of Microbiology and Immunology, Western University, London, ON, Canada.
Department of Cellular and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
Methods Mol Biol. 2022;2440:143-164. doi: 10.1007/978-1-0716-2051-9_9.
Understanding the interplay between commensals, pathogens, and immune cells in the skin and mucosal tissues is critical to improve prevention and treatment of a myriad of diseases. While high-parameter flow cytometry is the current gold standard for immune cell characterization in blood, it is less suitable for mucosal tissues, where structural and spatial information is lost during tissue disaggregation. Immunofluorescence overcomes this limitation, serving as an excellent alternative for studying immune cells in mucosal tissues. However, the use of immunofluorescent microscopy for analyzing clinical samples is hampered by a lack of high-throughput quantitative analysis techniques. In this chapter, we describe methods for sectioning, staining, and imaging whole sections of human foreskin tissue. We also describe methods to automate immune cell quantification from immunofluorescent images, including image preprocessing and methods to quantify both circular and irregularly shaped immune cells using open-source software.
了解共生菌、病原体和皮肤及黏膜组织中免疫细胞之间的相互作用，对于改善多种疾病的预防和治疗至关重要。虽然高参数流式细胞术是目前血液中免疫细胞特征分析的金标准，但它不太适用于黏膜组织，因为在组织分散过程中会丢失结构和空间信息。免疫荧光克服了这一限制，是研究黏膜组织中免疫细胞的绝佳替代方法。然而，免疫荧光显微镜在分析临床样本方面的应用受到缺乏高通量定量分析技术的限制。在本章中，我们描述了用于切割、染色和成像人体包皮组织全切片的方法。我们还描述了从免疫荧光图像中自动量化免疫细胞的方法，包括图像预处理以及使用开源软件量化圆形和非规则形状免疫细胞的方法。
