Abstract Deep insights into the complex cellular and molecular changes occurring during different (patho-)physiological conditions are essential for understanding the interactions and regulation of different proteins. This understanding is crucial for both research and diagnostics. However, the effectiveness of conventional immunofluorescence, an effective tool for visualizing the spatial distribution of cells or proteins, is limited in complex tissues. This is mainly due to challenges such as the spectral overlap of fluorophore wavelengths, a limited range of antibody types, and the inherent variability of samples. Multiplex immunofluorescence imaging offers a solution to these limitations by enabling precise localization of proteins and identification of different cell types in a single tissue sample. 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 to all researchers through commercially available reagents and open-access image analysis codes. 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 a significant 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.

A super-resolution compatible workflow for highly multiplexed immunofluorescence of routinely processed kidney tissue