In Situ Hybridization of Labeled RNA Probes to Fixed Parhyale hawaiensis Embryos
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3140, USA
- Department of Integrative Biology, University of California, Berkeley, CA 94720-3140, USA
- Howard Hughes Medical Institute, University of California, Berkeley, CA 94720-3140, USA
- ↵1Corresponding author (nipam{at}uclink.berkeley.edu)
INTRODUCTION
The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol describes in situ hybridization of fluorescein- or digoxigenin (DIG)-labeled RNA probes to fixed P. hawaiensis embryos. Standard techniques of molecular biology should be used to produce an appropriate template for generation of antisense RNA probes. RNA-labeling mixes designed to produce fluorescein- or DIG-labeled RNA probes using T3, T7, or SP6 RNA polymerases are commercially available. Probes should be purified using QIAGEN RNeasy columns or similar means. Considerations for double-labeling experiments using both fluorescein- and DIG-labeled RNA probes are included.
