Whole-mount in situ hybridization of Hawaiian bobtail squid (Euprymna scolopes) embryos with DIG-labeled riboprobes: II. Embryo preparation, hybridization, washes, and immunohistochemistry.
Whole-mount in situ hybridization is a technique used to localize and visualize specific gene transcripts in whole embryos by hybridizing labeled RNA probes complementary to the sequence of interest. A digoxigenin (DIG)-labeled riboprobe synthesized during in vitro transcription through the incorporation of DIG-labeled UTP is hybridized to the target sequence under stringent conditions, and excess unhybridized probe is removed during a series of washes. The location of the labeled riboprobe, and thus the mRNA sequence of interest, is then visualized by immunohistochemistry. This protocol outlines the steps involved in preparing Hawaiian bobtail squid (Euprymna scolopes) embryos, hybridizing a DIG-labeled riboprobe in whole-mount embryos, and visualizing the labeled RNA colorimetrically using an alkaline-phosphatase-conjugated anti-DIG antibody.
Department of Zoology, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
Patricia N
PN
Lee
Margaret J
MJ
McFall-Ngai
Patrick
P
Callaerts
H Gert
HG
de Couet
D000818:Animals; D049832:Decapodiformes / Q000196:embryology* / Q000235:genetics; D004076:Digoxigenin / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D006254:Hawaii; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D015347:RNA Probes / Q000235:genetics*; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation
D015347:RNA Probes; D004076:Digoxigenin