Imaging Cilia in Zebrafish

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Methods in cell biology (Impact Factor: 1.42). 01/2010; 97:415-35. DOI: 10.1016/S0091-679X(10)97022-2
Source: PubMed


Research focused on cilia as extremely important cellular organelles has flourished in recent years. A thorough understanding of cilia regulation and function is critical, as disruptions of cilia structure and/or function have been linked to numerous human diseases and disorders. The tropical freshwater zebrafish is an excellent model organism in which to study cilia structure and function. We can readily image cilia and their motility in embryonic structures including Kupffer's vesicle during somite stages and the pronephros from 1 day postfertilization onward. Here, we describe how to image cilia by whole-mount immunofluorescence, transverse cryosection/immunohistochemistry, and transmission electron microscopy. We also describe how to obtain videos of cilia motility in living embryos.

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    • "Embryos were prepared for flat-mounting by dissecting the yolk away from the embryo proper with a pair of fine forceps; the embryos were then mounted between two glass coverslips. Immunofluorescence detection of acetylated tubulin in the whole-mount embryos was performed according to a published protocol (Jaffe et al., 2010). High-speed video microscopy analysis has been described in detail in a recent publication (Pathak et al., 2011). "
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    • "Gene knockdown can be efficiently achieved with morpholino antisense oligonucleotides (with appropriate controls to verify specificity and detect off-target effects), so both forward and reverse genetic tools are available. Combined with high-quality in vivo imaging (Jaffe et al., 2010; Wallingford, 2010a, 2010b, 2010c), these features render frog and fish attractive models for the study of cilia in vertebrates. "
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