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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

Published: April 23rd, 2021

DOI:

10.3791/62079

1Department of Nephrology, Medical Faculty, Heinrich-Heine-University
* These authors contributed equally

The presented method enables visualization of fluorescently labeled cellular proteins with expansion microscopy leading to a resolution of 70 nm on a conventional microscope.

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte foot processes contain actin bundles that bind to cytoskeletal adaptor proteins such as podocin. Those adaptor proteins, such as podocin, link the backbone of the glomerular slit diaphragm, such as nephrin, to the actin cytoskeleton. Studying the localization and function of these and other podocytic proteins is essential for the understanding of the glomerular filter's role in health and disease. The presented protocol enables the user to visualize actin, podocin, and nephrin in cells with super resolution imaging on a conventional microscope. First, cells are stained with a conventional immunofluorescence technique. All proteins within the sample are then covalently anchored to a swellable hydrogel. Through digestion with proteinase K, structural proteins are cleaved allowing isotropical swelling of the gel in the last step. Dialysis of the sample in water results in a 4-4.5-fold expansion of the sample and the sample can be imaged via a conventional fluorescence microscope, rendering a potential resolution of 70 nm.

Albuminuria is a surrogate parameter of cardiovascular risk and results from disruption of the glomerular filter1. The glomerular filter is composed of the fenestrated endothelium, the glomerular basement membrane and the slit diaphragm formed by podocytes. Primary and secondary foot processes of podocytes wrap around the capillary wall of the glomerulum2. The delicate structure of foot processes is maintained by cortical actin bundles which also serve as anchors for multiple slit diaphragm proteins and other adaptor proteins2. The slit diaphragm's backbone protein is called nephrin and intera....

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1. Splitting and seeding of cells

  1. Warm up sterile Dulbecco's Modified Eagle's Medium (DMEM) including 10% fetal calf serum (FCS), sterile Phosphate buffered saline (PBS) and sterile trypsin to 37 °C. Activate the clean bench.
  2. Prepare a 6-well plate by adding one sterile glass cover slip (10 mm) to each well using sterile forceps.
  3. Put a 10 cm cell culture dish with Cos7 cells under the clean bench. Under the clean bench, aspirate the cells' medium using a vacuum device.

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The concept and timing of this proExM protocol is depicted in Figure 1. On day 5, transfected cells are fixed and stained with fluorescent antibodies targeting the protein of interest (Figure 1A,B). On day 6, treatment with AcX leads to formation of amine groups on all proteins (including fluorophores) (Figure 1A,B)12. Upon polymerization of t.......

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The presented method enables the investigator to visualize cellular proteins, e.g., podocin, nephrin, and cytoskeletal components, e.g., F-actin. Within this protocol, transfected cos7 cells are used as a model to study interaction of slit diaphragm proteins with F-actin. Unfortunately, immortalized podocyte cell lines do not express sufficient endogenous amounts of slit diaphragm proteins19.

With this method, cellular proteins can be visualized with nanoscale resolutio.......

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The authors would like to thank Blanka Duvnjak and Nikola Kuhr for their excellent technical assistance.

....

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Name Company Catalog Number Comments
Acrylamide >99% Sigma-Aldrich A3553-100G
6-((Acryloyl)amino)hexanoic acid, succinimidyl ester, Acryloyl-X, SE invitrogen A-20770 store up to 4 months
APS Sigma-Aldrich A3678-25G
Deckgläser (cover glasses) Engelbrecht K12432 24x32mm #1.0
Diamont cutter VWR 201-0392 for cutting the cover slips
Guanidine HCl Sigma-Aldrich G3272-100G 8M Stock can be kept at RT
Marten hair paintbrush Leon Hardy 3 (770)
"Menzel" Deckgläser (cover glasses) Thermo Fischer  15654786 24x24mm #1.5
N,N`-Methylenbisacrylamide Sigma-Aldrich M7256-25G
Objektträger UniMark Marienfeld 703010
Proteinase K New England Biolabs P8107S
Sodium Acrylate Sigma-Aldrich 408220 check purity 
Sodium Bicarbonate Sigma-Aldrich S5761
Staining chamber produced at the university's workshop
TEMED ROTH 2367.1
6-Well glass bottom plates Cellvis P06-1.5H-N
Antibodies
Actin-ExM 546  chrometra non-available 1:40
Anti Podocin produced in rabbit Sigma P-0372-200UL 1:200
Donkey anti guinea-pig CF633 Sigma SAB4600129-50UL 1:200
Goat anti rabbit 488 Life Technologies A11034 1:1000
Guinea pig anti nephrin Origene BP5030 1:100
Software
FIJI
Visiview
microscope
AXIO Observer Z1 Zeiss non-available

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