Published: April 23rd, 2021
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....
1. Splitting and seeding of cells
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.......
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.......
|6-((Acryloyl)amino)hexanoic acid, succinimidyl ester, Acryloyl-X, SE
|store up to 4 months
|Deckgläser (cover glasses)
|for cutting the cover slips
|8M Stock can be kept at RT
|Marten hair paintbrush
|"Menzel" Deckgläser (cover glasses)
|New England Biolabs
|produced at the university's workshop
|6-Well glass bottom plates
|Anti Podocin produced in rabbit
|Donkey anti guinea-pig CF633
|Goat anti rabbit 488
|Guinea pig anti nephrin
|AXIO Observer Z1
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