Live dissection of Drosophila embryos: streamlined methods for screening mutant collections by antibody staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such...

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Bibliographic Details
Published in:Journal of visualized experiments 2009-12 (34)
Main Authors: Lee, Hyung-Kook Peter, Wright, Ashley P, Zinn, Kai
Format: Article
Language:English
Subjects:
Animals
Antibodies - chemistry
Central Nervous System - embryology
Central Nervous System - surgery
Developmental Biology
Dissection - methods
Drosophila - embryology
Drosophila - genetics
Embryo, Nonmammalian - surgery
Mutation
Staining and Labeling - methods
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Summary:Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.
ISSN:1940-087X
1940-087X
DOI:10.3791/1647