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Abstract: PO2230

Ultra-Fast Clearing Protocol for 3D Optical Renal Pathology

Session Information

Category: Pathology and Lab Medicine

  • 1601 Pathology and Lab Medicine: Basic

Authors

  • Unnersjö-Jess, David, Science for life Laboratory, Dept. of Applied Physics, Royal Institute of Technology, Solna, Stockholm, Sweden
  • Butt, Linus, Department II of Internal Medicine and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Nordrhein-Westfahlen, Germany
  • Höhne, Martin, Department II of Internal Medicine and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Nordrhein-Westfahlen, Germany
  • Witasp, Anna, Karolinska Universitetsjukhuset i Huddinge, Huddinge, Sweden
  • Kuehne, Lucas, Department II of Internal Medicine and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Nordrhein-Westfahlen, Germany
  • Hoyer, Peter, Pediatric Nephrology, Pediatrics II, Univeristy of Duisburg-Essen, Essen, Nordrhein-Westfahlen, Germany
  • Patrakka, Jaakko, Karolinska Universitetsjukhuset i Huddinge, Huddinge, Sweden
  • Brinkkoetter, Paul T., Department II of Internal Medicine and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Nordrhein-Westfahlen, Germany
  • Wernerson, Annika, Karolinska Universitetsjukhuset i Huddinge, Huddinge, Sweden
  • Schermer, Bernhard, Department II of Internal Medicine and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Nordrhein-Westfahlen, Germany
  • Benzing, Thomas, Department II of Internal Medicine and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Nordrhein-Westfahlen, Germany
  • Scott, Lena, Science for Life Laboratory, Dept. of Women's and Children's Health, Karolinska Institutet, Solna, Sweden, Solna, Stockholm, Sweden
  • Brismar, Hjalmar, Science for Life Laboratory, Dept. of Women's and Children's Health, Karolinska Institutet, Solna, Sweden, Solna, Stockholm, Sweden
  • Blom, Hans, Science for life Laboratory, Dept. of Applied Physics, Royal Institute of Technology, Solna, Stockholm, Sweden
Background

Kidney pathology involves three separate microscopy workflows. Histology, typically carried out on paraffin-embedded sections, used to optically visualize large-scale renal morphology. Immunofluorescence, used for mapping presence of specific molecular variations, carried out on paraffin-embedded or fresh-frozen thin sections. To visualize renal ultrastructures, electron microscopy is applied, with elaborate preparation protocols on ultra-thin sections.

Methods

We have modified published protocols in order to simplify and speed up the workflow of routine optical kidney imaging. The presented protocol induces a slight swelling of a cleared sample, and increase effective resolution enough for 3-D confocal visualization of filtration barrier structures. The duration of the protocol is only 5 hours from harvesting the tissue until full image acquisition.

Results

Our simple and fast protocol can resolve foot processes in mouse and human tissue using standard lab equipment and conventional 3-D confocal microscopy. Importantly, the protocol can be used to visualize various large-scale histopathological features as well as immune deposits in human patient material too. Compared to others, our tissue protocol is simpler and faster, and allow better 3-D in situ imaging capabilities.

Conclusion

We conclude that our simple and fast protocol, allow researchers and pathologists to use a single preparation and microscopy technique, to visualize both renal ultrastructure, histology and protein expression. Our protocol has the potential of, not just to complement, but also merging workflows used today while adding accessing to in situ depth information on all scales.

Funding

  • Government Support - Non-U.S.