Super-resolved highly multiplexed immunofluorescence imaging for precise protein localization and podocyte ultrastructure.
kidney disease
multiplex immunofluorescence
super resolution microscopy
Journal
Journal of cellular and molecular medicine
ISSN: 1582-4934
Titre abrégé: J Cell Mol Med
Pays: England
ID NLM: 101083777
Informations de publication
Date de publication:
Sep 2024
Sep 2024
Historique:
revised:
19
07
2024
received:
29
04
2024
accepted:
03
08
2024
medline:
28
9
2024
pubmed:
28
9
2024
entrez:
28
9
2024
Statut:
ppublish
Résumé
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.
Substances chimiques
Cadherins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e70066Subventions
Organisme : Bundesministerium für Bildung und Forschung
ID : STOP-FSGS/01GM1901Band01GM2202B
Informations de copyright
© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.
Références
Park J, Shrestha R, Qiu C, et al. Single‐cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease. Science. 2018;360(6390):758‐763.
Chung JJ, Goldstein L, Chen YJJ, et al. Single‐cell transcriptome profiling of the kidney glomerulus identifies key cell types and reactions to injury. J Am Soc Nephrol. 2020;31(10):2341‐2354.
Zimmermann M, Klaus M, Wong MN, et al. Deep learning‐based molecular morphometrics for kidney biopsies. JCI Insight. 2021;6(7):e144779.
Siegerist F, Ribback S, Dombrowski F, et al. Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement. Sci Rep. 2017;7(1):11473.
Rajagopalan A, Venkatesh I, Aslam R, et al. SeqStain is an efficient method for multiplexed, spatialomic profiling of human and murine tissues. Cell Rep Methods. 2021;1(2):100006.
Gut G, Herrmann MD, Pelkmans L. Multiplexed protein maps link subcellular organization to cellular states. Science. 2018;361(6401):eaar7042.
Artelt N, Ludwig TA, Rogge H, et al. The role of palladin in podocytes. J Am Soc Nephrol. 2018;29(6):1662‐1678.
Hörl D, Rojas Rusak F, Preusser F, et al. BigStitcher: reconstructing high‐resolution image datasets of cleared and expanded samples. Nat Methods. 2019;16(9):870‐874.
Siegerist F, Hay E, Dikou JS, et al. ScoMorphoFISH: a deep learning enabled toolbox for single‐cell single‐mRNA quantification and correlative (ultra‐)morphometry. J Cell Mol Med. 2022;26(12):3513‐3526.
Artelt N, Siegerist F, Ritter AM, et al. Comparative analysis of podocyte foot process morphology in three species by 3D super‐resolution microscopy. Front Med. 2018;5:292.
Tesch F, Siegerist F, Hay E, et al. Super‐resolved local recruitment of CLDN5 to filtration slits implicates a direct relationship with podocyte foot process effacement. J Cell Mol Med. 2021;25(16):7631‐7641.
Pullman JM, Nylk J, Campbell EC, Gunn‐Moore FJ, Prystowsky MB, Dholakia K. Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease. Biomed Opt Express. 2016;7(2):302.
Schell C, Sabass B, Helmstaedter M, et al. ARP3 controls the podocyte architecture at the kidney filtration barrier. Dev Cell. 2018;47(6):741‐757.e8.
Wunderlich LCS, Ströhl F, Ströhl S, Vanderpoorten O, Mascheroni L, Kaminski CF. Superresolving the kidney—a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier. Anal Bioanal Chem. 2021;413(4):1203‐1214.
Siegerist F, Drenic V, Koppe T‐M, Telli N, Endlich N. Super‐resolution microscopy: a technique to revolutionize research and diagnosis of glomerulopathies. Glomerular Dis. 2023;3(1):19‐28.
Siegerist F, Endlich K, Endlich N. Novel microscopic techniques for podocyte research. Front Endocrinol (Lausanne). 2018;9:379.
Kylies D, Zimmermann M, Haas F, et al. Expansion‐enhanced super‐resolution radial fluctuations enable nanoscale molecular profiling of pathology specimens. Nat Nanotechnol. 2023;18(4):336‐342.
Laine RF, Tosheva KL, Gustafsson N, et al. NanoJ: a high‐performance open‐source super‐resolution microscopy toolbox. J Phys D Appl Phys. 2019;52(16):163001.
Shaib AH, Chouaib AA, Chowdhury R, et al. Visualizing proteins by expansion microscopy. bioRxiv. 2023;8(3):502284.
Kuppe C, Leuchtle K, Wagner A, et al. Novel parietal epithelial cell subpopulations contribute to focal segmental glomerulosclerosis and glomerular tip lesions. Kidney Int. 2019;96(1):80‐93.
Hansen KUI, Siegerist F, Daniel S, et al. Prolonged podocyte depletion in larval zebrafish resembles mammalian focal and segmental glomerulosclerosis. FASEB J. 2020;34(12):15961‐15974.
Hodgin JB, Borczuk AC, Nasr SH, et al. A molecular profile of focal segmental glomerulosclerosis from formalin‐fixed, paraffin‐embedded tissue. Am J Pathol. 2010;177(4):1674‐1686.