Balsini Parvaneh, Weninger Wolfgang, Pfisterer Karin
Department of Dermatology, Medical University of Vienna.
Department of Dermatology, Medical University of Vienna;
J Vis Exp. 2025 Aug 8(222). doi: 10.3791/68746.
The spatial distribution of immune and non-immune cells within tissues and the expression of cell-specific markers provides essential information on cell function in situ. Human skin is a highly complex organ with defined compartments comprising different cells with diverse phenotypes. By using multi-fluorescence labeling, distinct skin cell populations can be determined and further characterized. However, currently, the availability of non-commercial tools for image analysis of skin samples that allow in silico segmentation of cells specifically within the epidermis or the dermis at a single-cell resolution is limited. We provide here a step-by-step protocol for immunofluorescence staining of skin sections, confocal microscopy, and image analysis using our freely available tool SCAnED (Skin Compartment Analysis of Epidermis and Dermis). The SCAnED macro allows the precise identification and classification of cells and nuclei in both the epidermal and dermal compartments of human skin. We provide guidelines on how to determine average intensity levels of maker expression in different cells and cellular compartments, such as the cytoplasm and the nucleus, and how to quantify cells expressing varying amounts of these markers with an accompanying Python pipeline provided as a ready-to-use Jupyter notebook executed in Google Colab. This protocol will allow inexperienced users to determine cell-specific expression profiles within skin tissue and provide insights into the spatial distribution of cells within the epidermal and dermal compartments, allowing a deeper understanding of the intricate tissue structure and cell composition of human skin.
组织内免疫细胞和非免疫细胞的空间分布以及细胞特异性标志物的表达提供了关于原位细胞功能的重要信息。人类皮肤是一个高度复杂的器官，具有明确的分区，包含不同表型的不同细胞。通过使用多荧光标记，可以确定并进一步表征不同的皮肤细胞群体。然而，目前用于皮肤样本图像分析的非商业工具有限，这些工具能够以单细胞分辨率在计算机上对表皮或真皮内的细胞进行特异性分割。我们在此提供了一个分步方案，用于皮肤切片的免疫荧光染色、共聚焦显微镜检查以及使用我们免费提供的工具SCAnED（表皮和真皮皮肤分区分析）进行图像分析。SCAnED宏允许精确识别和分类人类皮肤表皮和真皮分区中的细胞和细胞核。我们提供了有关如何确定不同细胞和细胞区室（如细胞质和细胞核）中标志物表达平均强度水平的指南，以及如何使用作为在Google Colab中执行的即用型Jupyter笔记本提供的配套Python管道对表达不同量这些标志物的细胞进行定量的指南。该方案将使缺乏经验的用户能够确定皮肤组织内细胞特异性表达谱，并深入了解表皮和真皮分区内细胞的空间分布，从而更深入地了解人类皮肤复杂的组织结构和细胞组成。
