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

Eva Königshausen, Christina Theresa Schmitz, Lars Christian Rump, Lorenz Sellin

Journal of visualized experiments : JoVE 2021 Apr 23

filter terms:

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.

Citation

Eva Königshausen, Christina Theresa Schmitz, Lars Christian Rump, Lorenz Sellin. Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy. Journal of visualized experiments : JoVE. 2021 Apr 23(170)