This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.
Before you start
Immunohistochemistry
Mounting
Note : Mount in glycerol if samples will be imaged immediately. Mount in Prolong Gold if samples will be stored before imaging (for more than a week) or if samples must be imaged multiple times. To use, place a bottle of prolong in 65ºC water bath for 2-3 minutes. Take an aliquot of Prolong Gold and keep in on a heat-block at 45-50º C.
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 of Drosophila has gained great popularity since the early studies that illuminated its basic structure and function.1-4 Many of the molecules that have been identified in studies of the Drosophila NMJ are conserved in vertebrates.4 Therefore, the insights learned through studies of the Drosophila NMJ may be applicable to synaptic biology in many systems. Some possible applications include the study of molecules involved in synaptic development, plasticity, and neurological disease.
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 |