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Amphioxus Whole-Mount In Situ Hybridization
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</ul> </div> <div class="bar"> <div class="bar-inner"></div> </div> </div> <div id="content-block"> <div xmlns="" class="section-name protocol">Protocol</div> <div class="article abstract-view" itemprop="articleBody"><span class="highwire-journal-article-marker-start"></span><h1 id="article-title-1" itemprop="headline">Amphioxus Whole-Mount In Situ Hybridization</h1> <div class="contributors"> <ol class="contributor-list" id="contrib-group-1"> <li class="contributor" id="contrib-1" itemprop="author" itemscope="itemscope" itemtype="http://schema.org/Person"><span class="name" itemprop="name"><a class="name-search" href="/search?author1=J.K.+Sky+Yu&sortspec=date&submit=Submit">J.K. Sky Yu</a></span><a id="xref-aff-1-1" class="xref-aff" href="#aff-1">1</a><span class="xref-sep">,</span><a id="xref-corresp-1-1" class="xref-corresp" href="#corresp-1">3</a> and </li> <li class="last" id="contrib-2"><span class="name"><a class="name-search" href="/search?author1=Linda+Z.+Holland&sortspec=date&submit=Submit">Linda Z. Holland</a></span><a id="xref-aff-2-1" class="xref-aff" href="#aff-2">2</a></li> </ol> <ol class="affiliation-list"> <li class="aff"><a id="aff-1" name="aff-1"></a><address><sup>1</sup> Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China </address> </li> <li class="aff"><a id="aff-2" name="aff-2"></a><address><sup>2</sup> Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92037, USA </address> </li> </ol> <ol class="corresp-list"> <li class="corresp" id="corresp-1"><a class="rev-xref" href="#xref-corresp-1-1">↵</a><sup>3</sup>Corresponding author (<span class="em-link"><span class="em-addr">jkyu{at}gate.sinica.edu.tw</span></span>). </li> </ol> </div> <div class="section abstract" id="abstract-1" itemprop="description"> <h2>INTRODUCTION</h2> <p id="p-1">The in situ hybridization (ISH) technique is one of the basic methods of developmental biology. It allows the distributions of specific transcripts to be detected in fixed cells or embryos during development. This protocol describes ISH of digoxigenin (DIG)-labeled antisense RNA probes to whole-mount amphioxus embryos and larvae. Embryos are fixed and permeabilized before being hybridized with the DIG-labeled antisense RNA probes. Following hybridization, transcripts are detected immunohistochemically with an anti-DIG antibody conjugated to alkaline phosphatase. The gene expression patterns are visualized in the embryos with colorimeric substrates of alkaline phosphatase. Labeled embryos are photographed as whole mounts, after which they can be embedded in Spurr’s resin and sectioned if desired. To facilitate the handling of small amphioxus embryos during whole-mount ISH, we developed a method of solution exchange using homemade mesh-bottom baskets and multiwell plates. This method speeds up time-consuming aspiration and decanting of buffers and avoids accidental loss of precious embryos during these steps. 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