Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia.
School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.
Methods Mol Biol. 2024;2779:407-423. doi: 10.1007/978-1-0716-3738-8_19.
The complexities and cellular heterogeneity associated with tissues necessitate the concurrent detection of markers beyond the limitations of conventional imaging approaches in order to spatially resolve the relationships between immune cell populations and their environments. This is a necessary complement to single-cell suspension-based methods to inform a better understanding of the events that may underlie pathological conditions. Imaging mass cytometry is a high-dimensional imaging modality that allows for the concurrent detection of up to 40 protein markers of interest across tissues at subcellular resolution. Here, we present an optimized staining protocol for imaging mass cytometry with modifications that integrate RNAscope. This unique addition enables combined protein and single-molecule RNA detection, thereby expanding the utility of imaging mass cytometry to researchers investigating low abundance or noncoding targets. In general, the procedure described is broadly applicable for comprehensive immune profiling of host-pathogen interactions, tumor microenvironments and inflammatory conditions, all within the tissue contexture.
与组织相关的复杂性和细胞异质性需要超越传统成像方法的限制，同时检测标记物，以便在空间上解析免疫细胞群体与其环境之间的关系。这是对基于单细胞悬液的方法的必要补充，有助于更好地了解可能导致病理状况的事件。成像质谱细胞术是一种高维成像模式，允许在亚细胞分辨率下同时检测多达 40 种感兴趣的蛋白质标记物。在这里，我们提出了一种优化的成像质谱细胞术染色方案，其中包括 RNAscope 的修改。这种独特的添加使蛋白质和单分子 RNA 的联合检测成为可能，从而将成像质谱细胞术的用途扩展到研究低丰度或非编码靶标的研究人员。一般来说，所描述的程序广泛适用于宿主-病原体相互作用、肿瘤微环境和炎症条件的全面免疫分析，所有这些都在组织结构中进行。
