We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.
The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudière et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.
1. Preparation of Reagents
2. Collection of Embryos
3. Removal of the Chorion and Vitelline Membranes and Fixation
4. Antibody Staining, Mounting, and Visualization
5. Representative Results
CSP
HRP-FITC
Merged
Figure 1: A Representative images showing the embryonic CNS at stage 16-17, using antibodies against CSP (red) and HRP (green). Note the distinct staining of the commissure.
CSP
HRP-FITC
Merged
Figure 2: A representative images showing the embryonic PNS at stage 16-17 using antibodies against CSP (red) and HRP (green). Note the repeated patterns of peripheral neurons.
The Drosophila embryo is a powerful system for investigating the cellular and molecular changes during neuronal development. In this protocol we have demonstrated how to prepare whole mount embryos for immunohistochemistry. We have adapted this protocol from the protocol described in Carroll and Scott, 1985. This protocol can also be adapted to test other neuronal antibodies, but optimization of antibodies should be done. Further, developmental defects of the embryonic PNS and CNS can be easily visualized by comparing embryos from different mutant lines. We have successfully used this protocol to visualize the PNS and CNS in embryos carrying motor protein mutants. Under high magnification we have also evaluated the axonal tracks within the embryonic PNS and CNS.
The authors have nothing to disclose.
SG is supported by funds from the State University of New York at Buffalo and from John R. Oishei Foundation.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
37% formaldehyde | Fisher | BP531-25 | ||
CSP | Developmental Studies Hybridoma Bank | |||
HRP-FITC | Jackson ImmunoResearch Laboratories | 323-095-021 | ||
Alexa Fluor 568 Goat Anti-Mouse IgG | Invitrogen | A-11004 | ||
Vectashield Mounting Medium | Vector Laboratories, Inc. | H-1000 |