Article

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

ABSTRACT

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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    • "Regardless of technique—mRNA, morpholino, or peptide nucleic acid injection—the primary purpose is to demonstrate the appropriate change in protein level. If an antibody is available for the protein of interest, for determination of morpholino efficacy either a western blot from zebrafish protein fry extracts or whole mount immunohistochemistry staining can be performed [39] [40]. If an antibody is unavailable, a transgenic or coinjected mRNA with 5 í® í° UTR of the gene of interest upstream of a marker or epitope tag (e.g., hemagglutinin or GFP) could be used to assess the level of knockdown. "
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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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