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Kidney Week

Abstract: FR-OR100

Iterative Indirect Immunofluorescence Imaging for Tissue (4iT): The First Step Towards Spatial Proteomic Maps

Session Information

Category: Pathology and Lab Medicine

  • 1601 Pathology and Lab Medicine: Basic


  • Puelles, Victor G., University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Gernhold, Lukas, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Sarabia del Castillo, Jacobo, University of Zurich, Zurich, Switzerland
  • Meyer-Schwesinger, Catherine, University of Hamburg, Hamburg, Germany
  • Gnirck, Ann-Christin, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Braun, Fabian, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Turner, Jan-Eric, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Pelkmans, Lucas, University of Zurich, Zurich, Switzerland
  • Huber, Tobias B., University Medical Center Hamburg-Eppendorf, Hamburg, Germany

With the rapid development of multi-omics techniques, there is an increasing need for methods that can localize proteins with high spatial resolution. This is particularly important in a complex system like the kidney; where a large number of interacting cell-types coexist.


We present 4iT, a new method for highly multiplexed protein measurements in paraffin-embedded tissue based on the principle of cyclic immunolabelling, including conventional indirect immunofluorescence, high-resolution fluorescence imaging, and elusion of primary and secondary antibodies. Five glomeruli and periglomerular spaces were serially imaged per mouse (n=4 controls and n=8 crescentic glomerulonephritis) for a total of 40 cycles (1 antibody per cycle). Multiplexed protein maps were generated using an in-house unsupervised integration tool based on artificial neural networks that identified shared and unique pixel profiles.


We successfully identified endothelial cells, mesangial cells, basement membranes, podocytes, parietal epithelial cells, proximal tubuli, collecting ducts, immune cells, fibroblasts, innervation, subcellular units (ie. lysosomes, ER and mitochondria), and dynamic changes of extracellular matrix, integrins, tight-junctions, extra/intracellular receptors, phosphorylation sites, cell activation, and post-translational modifications. Multiplexed protein maps generated functional/pathological clusters based on proximity profiles.


4iT can be used to identify functionally relevant single-cell states in paraffin-embedded tissue, providing a new roadmap for molecular tissue analysis with high spatial resolution, and thereby personalized medicine.

4iT generates complex proteomic maps with high spatial resolution. (A) Example of 5 cycles with a total of 10 proteins identified in different kidney cells - first row shows stainings and second row shows the same glomerulus after elusion. (B) Proteomic map after deep learning and cluster/proximity analyses.