Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany.
Department for Pediatrics, University Medicine Greifswald, Greifswald, Germany.
J Cell Mol Med. 2024 Sep;28(18):e70066. doi: 10.1111/jcmm.70066.
Deep insights into the complex cellular and molecular changes occurring during (patho-)physiological conditions are essential for understanding the interactions and regulation of proteins. This understanding is crucial for research and diagnostics. However, the effectiveness of conventional immunofluorescence and light microscope, tools for visualizing the spatial distribution of cells or proteins, are limited both in resolution and multiplexity in complex tissues. This is mainly due to challenges such as the spectral overlap of fluorophore wavelengths, a limited range of antibody types, the inherent variability of samples and the optical resolution limit. The herein demonstrated combination of multiplex immunofluorescence imaging and super resolution microscopy offers a solution to these limitations by enabling the identification of different cell types and precise subcellular localization of proteins in tissue sections. In this study, we demonstrate the cyclic staining and de-staining of paraffin kidney sections, making it suitable for routine use and compatible with super-resolution microscopy for podocyte ultrastructural studies. We have further developed a computerized workflow for data processing which is accessible through available reagents and open-access code. As a proof of principle, we identified CDH2 as a marker for cellular lesions of sclerotic glomeruli in the nephrotoxic serum nephritis mouse model and cross-validated this finding with a human Nephroseq dataset indicating its translatability. In summary, our work represents an advance in multiplex imaging, which is crucial for understanding the localization of numerous proteins in a single FFPE kidney section and the compatibility with super-resolution microscopy to study ultrastructural changes of podocytes.
深入了解（病理）生理条件下发生的复杂细胞和分子变化对于理解蛋白质的相互作用和调节至关重要。这种理解对于研究和诊断至关重要。然而，传统免疫荧光和光学显微镜等用于可视化细胞或蛋白质空间分布的工具，在分辨率和复杂组织中的多重性方面都受到限制。这主要是由于荧光团波长的光谱重叠、抗体类型有限、样本固有变异性和光学分辨率限制等挑战所致。本文中展示的多重免疫荧光成像和超分辨率显微镜的组合通过能够识别不同的细胞类型和蛋白质在组织切片中的精确亚细胞定位，为这些限制提供了解决方案。在这项研究中，我们演示了石蜡肾切片的循环染色和去染色，使其适合常规使用，并与超分辨率显微镜兼容，用于足细胞超微结构研究。我们进一步开发了一种计算机化的数据处理工作流程，该流程可通过可用试剂和开放获取代码访问。作为原理验证，我们确定了 CDH2 作为肾毒性血清肾炎小鼠模型中硬化肾小球细胞病变的标志物，并通过人类 Nephroseq 数据集交叉验证了这一发现，表明其具有可转移性。总之，我们的工作代表了多重成像的进步，这对于理解单个 FFPE 肾切片中许多蛋白质的定位以及与超分辨率显微镜兼容以研究足细胞的超微结构变化至关重要。
