Department of Nephrology, Medical Faculty, Heinrich-Heine-University.
Department of Nephrology, Medical Faculty, Heinrich-Heine-University;
J Vis Exp. 2021 Apr 23(170). doi: 10.3791/62079.
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.
肾小球滤过膜由肾小球内皮细胞、肾小球基底膜和足细胞组成，滤过膜的破坏导致白蛋白尿。足细胞的足突含有肌动蛋白束，这些肌动蛋白束与细胞骨架衔接蛋白（如足细胞蛋白）结合。这些衔接蛋白，如足细胞蛋白，将肾小球裂孔隔膜的主干（如nephrin）与肌动蛋白细胞骨架连接起来。研究这些和其他足细胞蛋白的定位和功能对于理解肾小球滤过在健康和疾病中的作用至关重要。本方案通过在常规显微镜上进行超分辨率成像，使用户能够可视化细胞中的肌动蛋白、足细胞蛋白和nephrin。首先，通过常规免疫荧光技术对细胞进行染色。然后，样品中的所有蛋白质都被共价锚定到可溶胀的水凝胶上。通过蛋白酶 K 的消化，结构蛋白被切割，使得凝胶在最后一步进行各向同性膨胀。通过在水中对样品进行透析，样品的体积扩大了 4-4.5 倍，然后可以通过常规荧光显微镜对样品进行成像，得到 70nm 的潜在分辨率。
