Whole-mount in situ hybridization in monodelphis embryos

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. This protocol details whole-mount in situ hybridization of Monodelphis embryos, but it is broadly applicable to any marsupial. Special conditions have been included throughout the protocol for various stages of marsupial embryos. Nevertheless, whole, preterm embryonic stages (~stage 33 to birth) have proven to be difficult to work with because formation of the cuticle prevents probe and antibody penetration.