Summary
This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.
Abstract
The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.
Protocol
Before you start Prepare the following solutions: PBT (0.1 % Triton X100 in 1X PBS), PBTB ( 0.2% BSA in PBT), and PBTN (2% NGS in PBTB). Dissect Drosophila larvae. Please see Drosophila Larval NMJ Dissection. Immunohistochemistry Move the larvae to a 1.5 ml tube containing PBT. Wash the larvae twice for 15 minutes in the PBT. Note: to wash, place the 1.5 ml tube on a nutator mixer. Remove the liquid with a pippeto…
Discussion
Immunohistochemistry (IHC) is vital for the study of NMJ biology because it enables visualization of the NMJ. This is accomplished by using antibodies that recognize the neuronal membrane (e.g., HRP), the presynapse (e.g., CSP, SYT), and/or the postsynapse (e.g., DLG). Signaling molecules, structural proteins, or novel proteins of interest can also be stained. Then, genes can be mutated or missexpressed, and IHC can detect perturbation of synaptic structure and/or neuronal signaling.
The NMJ o…
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Stereomicroscope “Stemi” 2000 | Carl Zeiss MicroImaging Inc. | 495101-9804-000 | ||
| Light Source KL 1500 LCD | Carl Zeiss MicroImaging Inc. | 000000-1063-181 | ||
| Dumont SS Forceps | Fine Science Tools Inc. | 11200-33 | ||
| Dumont #5 Forceps | Fine Science Tools Inc. | 11252-20 | ||
| Adams™ Nutator Mixer | Becton, Dickinson and Company | 421105 | ||
| No.1 Precision knife | X-Acto | 3201 | ||
| No. 16 Blades | X-Acto | 216 | ||
| ProLong Gold Antifade reagent | Invitrogen Corporation | 36930 |
References
- Jan, L. Y., Jan, Y. N. Properties of the larval neuromuscular junction in Drosophila Melanogaster. J. Physiol. 262, 189-214 (1976).
- Johansen, J., Halpern, M. E., Johansen, K. M., Keshishian, H. Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae. J Neurosci. 9, 710-725 (1989).
- Kesheshian, H., Broadie, K., Chiba, A., Bate, M. The Drosophila neuromuscular junction: a model system for studying synaptic development and function. Annu Rev Neurosci. 19, 545-575 (1996).
- Collins, C. A., DiAntonio, A. Synaptic development: insights from Drosophila. Curr Opin Neurobiol. 17 (1), 35-42 (2007).
Citer Cet Article
Brent, J., Werner, K., McCabe, B. D. Drosophila Larval NMJ Immunohistochemistry. J. Vis. Exp. (25), e1108, doi:10.3791/1108 (2009).