Light-Sheet Fluorescence Microscopy Imaging of Whole-Mount Immunostained Mouse Embryos and Limbs

Animal development is the complex process that enables the formation of a pluricellular organism starting from a fertilized zygote. During this process, different tissues need to grow and differentiate in a coordinated manner in order to generate a fully functional organism. Oftentimes, technical or experimental difficulties cause imaging studies to focus specifically on the development of a specific tissue as if it were independent of the development of closely related structures. Further, classical analyses are performed on histological sections in two dimensions, which often only cover part of the tissue or organ under investigation. These analyses thus miss important information that can only be revealed in three-dimensional (3D) settings, which also allow the study of complete large structures. Here, we describe a method to perform whole-mount immunostaining and light-sheet fluorescence microscopy imaging of whole embryos and limbs up to the neonatal stage. While we focus on the co-development of skeletal muscle, tendons, and nerves, these techniques can be adapted to obtain high-resolution 3D images of large samples for the study of any other biological process.