A rapid protocol for whole-mount in situ hybridization on Xenopus embryos

INTRODUCTIONThis in situ hybridization (ISH) protocol describes a simplified method using a digoxigenin-labeled antisense RNA probe on whole Xenopus embryos, suitable for both X. laevis and X. tropicalis. The protocol includes fixation, β-galactosidase staining (when lineage tracing is needed), and storage of the embryos prior to ISH. This method shortens the steps before hybridization, which limits RNA degradation in the sample, and preserves superficial structures. Hence, it is particularly suited for the analysis of ectoderm, neural, and mesodermal structures from blastula to early tadpole stages. Additional permeabilization steps are included to process later tadpole stages.