Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5130

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In Situ Hybridization of Labeled RNA Probes to Fixed Parhyale hawaiensis Embryos

E. Jay Rehm, Roberta L. Hannibal, R. Crystal Chaw, Mario A. Vargas-Vila, and Nipam H. Patel1

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)

This article is also available in Emerging Model Organisms: A Laboratory Manual, Vol. 1. CSHL Press, Cold Spring Harbor, NY, USA, 2009.


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.


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The Crustacean Parhyale hawaiensis: A New Model for Arthropod Development
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E. J. Rehm, R. L. Hannibal, R. C. Chaw, M. A. Vargas-Vila, and N. H. Patel
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