Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

Summary

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Abstract

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.

Protocol

1. Preparation of Reagents

1. Prepare the PEMFA Buffer using 100mM Pipes, 2mM EGTA, and 1 mM MgSO₄. Adjust the pH of the buffer to 7.0. The PEMFA buffer is filter sterilized and stored at 4°C.
2. Prepare PBT by adding 1ml of Triton X-100 to 100ml 1x PBS (Phosphate Buffered Saline) (pH 7.0). Store at 4°C.
3. Prepare 5% BSA (bovine serum albumin) with 0.01% NaAcide in 1x PBS (pH 7.0).
4. Prepare the saline solution using 0.7% NaCl and 0.03% Triton X-100 in deionized water.
5. Prepare the fixative by combining 4 parts PEMFA buffer with 1 part 37% Formaldehyde.

2. Collection of Emb....

Discussion

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 com.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
37% formaldehyde	Fisher Scientific	BP531-25
CSP	Developmental Studies Hybridoma Bank
HRP-FITC	Jackson ImmunoResearch	323-095-021
Alexa Fluor 568 Goat Anti-Mouse IgG	Invitrogen	A-11004
Vectashield Mounting Medium	Vector Laboratories	H-1000