In situ hybridization of labeled RNA probes to fixed Parhyale hawaiensis embryos.
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.
Rehm
E Jay
EJ
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3140, USA.
Hannibal
Roberta L
RL
Chaw
R Crystal
RC
Vargas-Vila
Mario A
MA
Patel
Nipam H
NH
D033304:Amphipoda / Q000502:physiology*; D000818:Animals; D015509:Developmental Biology / Q000379:methods*; D004076:Digoxigenin / Q000494:pharmacology; D004625:Embryo, Nonmammalian / Q000196:embryology*; D019793:Fluorescein / Q000494:pharmacology; D017403:In Situ Hybridization / Q000379:methods*; D016376:Oligonucleotides, Antisense / Q000235:genetics; D015347:RNA Probes / Q000235:genetics*
D016376:Oligonucleotides, Antisense; D015347:RNA Probes; D004076:Digoxigenin; D019793:Fluorescein