Protocol

Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos with DIG-Labeled Riboprobes: I. DNA Template Preparation and In Vitro Transcription of Riboprobes

¹ Department of Zoology, University of Hawaii at Manoa, Honolulu, HI 96822, USA
² Kewalo Marine Laboratory, Pacific Biosciences Research Center/University of Hawaii at Manoa, Honolulu, HI 96813, USA
³ Department of Medical Microbiology, University of Wisconsin-Madison, Madison, WI 53706-1521, USA
⁴ Laboratory of Developmental Genetics, K.U. Leuven and VIB, B-3000 Leuven, Belgium

⁵Corresponding author (couet{at}hawaii.edu).

INTRODUCTION

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 a 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 techniques for preparing RNA probes for whole-mount in situ hybridization in Hawaiian bobtail squid (Euprymna scolopes) embryos from linearized plasmid DNA or polymerase chain reaction (PCR) products.