Methods for Visualization of RNA and Cytoskeletal Elements in the Early Zebrafish Embryo

Zebrafish embryos, with their large size (>0.5 mm) and accessibility, are valuable tools for investigating core cellular processes. Many of those processes, such as cell division, asymmetric inheritance of cellular components, and structural dynamics involved in cell motility and morphology rely on cytoskeletal rearrangements and associated macromolecules. In addition to the protein-rich cytoskeleton, the early embryo is packed with maternally deposited RNA, which serves essential roles in establishing cell polarity, cell fate, and cell organization. Here, we present methods for visualizing endogenous RNA along with cytoskeletal structures, including microtubules and filamentous actin (F-actin) in the context of an intact vertebrate embryo. Each of the four protocols described herein (embryo fixation, RNA probe design/synthesis, double fluorescent in situ hybridization with tubulin immunofluorescence, and fluorescent in situ hybridization with phalloidin labeling of F-actin) are intended for optimal preservation and visualization of both the cytoskeleton and RNAs of interest. These methods can also be modified and applied to a broad range of other uses.