Organ Culture and Immunostaining of Mouse Embryonic Kidneys

Abstract

INTRODUCTIONThe study of organogenesis in mammals allows investigation of a wide variety of basic cell biological processes in the context of the intact organ. This has become especially important in the age of genetics, as the consequences of gene deletion or mutation in the mouse can be directly linked to human congenital abnormalities. The ability to culture some organs ex vivo during development has emerged as an important tool to understand how tissues are constructed and the signaling pathways that regulate these processes. It has been especially useful in organs that grow via branching morphogenic mechanisms, such as the lung and kidney. Here we demonstrate isolation, ex vivo growth, and fluorescent immunostaining of mouse embryonic day 12.5 (E12.5) kidneys. To demonstrate nephron formation using live imaging, we have isolated and cultured kidneys from mice carrying a green fluorescent protein (GFP) transgene driven by the Hes1 promoter, which is expressed early in the developing nephron. We also provide a protocol for robust imaging of multiple kidney structures in the whole-mount setting. These techniques serve as a basic platform for the analysis of branching morphogenesis and nephron formation in genetic mouse models or in response to exogenous factors, such as agonists or inhibitors, which can be directly added to the culture medium.