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Abstract: PO1803

SeqStain: A Multiplex Staining Method for Deep Profiling of Human Kidney Tissues

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Rajagopalan, Anugraha R., Drug Discovery Center, Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois, United States
  • Venkatesh, Ishwarya, Drug Discovery Center, Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois, United States
  • Aslam, Rabail, Drug Discovery Center, Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois, United States
  • Gupta, Vineet, Drug Discovery Center, Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois, United States
Background

Chronic Kidney Disease (CKD) is an emerging global health challenge, affecting 10-15% of the population. Lack of reliable biomarkers precludes the early diagnosis of CKD. The progression of CKD is known to be dictated by renal tissue changes. For example, in the case of Diabetic Nephropathy, disease progression is accompanied by considerable changes to the glomeruli including the thickening of the glomerular basement membrane and mesangial expansion, extracellular matrix accumulation, reduced podocyte number, inflammation of the renal tissue, the influx of immune cells which ultimately lead to tissue damage and progression to CKD. Understanding these tissue-centered events on a deeper level is imperative to reduce morbidity associated with CKD and for early diagnosis.

Methods

To aid high-level multiplex staining of these tissues by immunofluorescence, we developed a novel multiplex staining method called SeqStain. The SeqStain multiplex platform uses DNA tagged antibodies and Fab fragments to stain while endonucleases are used to achieve gentle de-staining after each round. We designed a SeqStain multiplex panel with antibodies to probe different histological regions relevant to the kidney. Antibodies or Fab fragments were tagged with DNA oligonucleotide duplex which carries multiple fluorophores. Labeled Fab fragments were pre-complexed with primary antibodies for staining.

Results

SeqStain modified antibodies and Fab fragments efficiently labeled multiple markers in tissue sections. Kidney tissues were stained with the SeqStain reagents and de-stained using endonucleases and provided a simple, gentle, and rapid technique for multiplex imaging of the tissues. The method was implemented using a custom flow chamber and allowed the labeling of tens of antigens on a single tissue section. Image alignment and analyses provided spatialomic data on multiple cell types in the tissue.

Conclusion

SeqStain method offers a robust yet gentle multiplex staining method to profile the CKD kidney tissues and comprehend the tissue-centered events that could play a role in the disease progression. Currently, we are profiling the CKD tissues in multiplex staining experiments and comparing it to healthy human kidney tissues to generate the spatial maps.

Funding

  • Other NIH Support