Name: drosophila larval nmj immunohistochemistry
Uploaded: 2009-03-28T00:00:00
Description: Watch this Scientific Journal Video about Drosophila Larval NMJ Immunohistochemistry at JoVE.com

Before you start
<ol>
<li>Prepare the following solutions: PBT (0.1 % Triton X100 in 1X PBS), PBTB ( 0.2% BSA in PBT), and PBTN (2% NGS in PBTB).</li>
<li>Dissect Drosophila larvae. Please see Drosophila Larval NMJ Dissection.</li>
</ol>
Immunohistochemistry
<ol>
<li>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...

<ol>
	<li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+larval+neuromuscular+junction+in+Drosophila+Melanogaster.">Properties of the larval neuromuscular junction in Drosophila Melanogaster.</a> J. Physiol. 262, 189-214 (1976).</li><li>Johansen, J., Halpern, M. E., Johansen, K. M., Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotypic+morphology+of+glutamatergic+synapses+on+identified+muscle+cells+of+Drosophila+larvae.">Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae.</a> J Neurosci. 9, 710-725 (1989).</li><li>Kesheshian, H., Broadie, K., Chiba, A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+neuromuscular+junction:+a+model+system+for+studying+synaptic+development+and+function.">The Drosophila neuromuscular junction: a model system for studying synaptic development and function.</a> Annu Rev Neurosci. 19, 545-575 (1996).</li><li>Collins, C. A., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+development:+insights+from+Drosophila.">Synaptic development: insights from Drosophila.</a> Curr Opin Neurobiol. 17 (1), 35-42 (2007).</li></ol>

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...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stereomicroscope &#x201C;Stemi&#x201D; 2000</td>
<td>&nbsp;</td>
<td>Carl Zeiss MicroImaging Inc.</td>
<td>495101-9804-000</td>
<td>&nbsp;</td>
<tr>
<td>Light Source KL 1500 LCD</td>
<td>&nbsp;</td>
<td>Carl Zeiss MicroImaging Inc. </td>
<td>000000-1063-181</td>
<td>&nbsp;</td>
<tr>
<td>Dumont SS Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools Inc.</td>
<td>11200-33</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools Inc.</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Adams&#x2122; Nutator Mixer</td>
<td>&nbsp;</td>
<td>Becton, Dickinson and Company</td>
<td>421105</td>
<td>&nbsp;</td>
<tr>
<td>No.1 Precision knife</td>
<td>&nbsp;</td>
<td>X-Acto</td>
<td>3201</td>
<td>&nbsp;</td>
<tr>
<td>No. 16 Blades</td>
<td>&nbsp;</td>
<td>X-Acto</td>
<td>216</td>
<td>&nbsp;</td>
<tr>
<td>ProLong Gold Antifade reagent</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>36930</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

drosophila larval nmj immunohistochemistry

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.

Watch this Scientific Journal Video about Drosophila Larval NMJ Immunohistochemistry at JoVE.com

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Research

JoVE Journal

Biology

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Drosophila Larval NMJ Dissection

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Dissection and Staining of Drosophila Larval Ovaries

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Drosophila Pupal Abdomen Immunohistochemistry

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...