Ctenophore whole-mount antibody staining.

INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. Because of the ease of embryo collection, their size (up to 1 mm in some species), and their rapid development, ctenophores have been attractive animals for experimental embryologists. This protocol describes how to fix ctenophore embryos and their cydippid larvae for antibody staining. Once the samples have been fixed, tissues are incubated with an antibody to the epitope of interest. A secondary antibody conjugated to a fluorescent molecule then reveals the expression pattern of the epitope. Fluorescent microscopy is used to visualize and document the signal. The protocol also includes methods for staining or counterstaining with a fluorescent derivative of phalloidin, which reveals F-actin in muscles and cell borders. Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.