Name: immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall
Uploaded: 2011-11-10T13:00:00
Description: Watch this Scientific Journal Video about Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall at JoVE.com

We thank RIKEN for funding. P10-Gal4 was a kind gift of Alain Vincent (Universit&eacute; Paul Sabatier, Toulouse, France).

1. Preparation of reagents
Notes before beginning: Dissection and immunohistochemical staining are carried out in a magnetic chamber and the larva is pinned down using specially shaped insect pins. Detailed instructions on the construction of a magnetic chamber, and preparation of these pins can be found in related references 14,15. In brief, a 1x1cm square hole is cut into a magnetic sheet and a coverslip affixed to the rear of the sheet to make a small chamber. The ...

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To understand how complex cell shapes are achieved it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to assay microtubule organization of dendritic arborization neuron sensory dendrites. In addition to staining sensory neurons, this method achieves robust immunohistological staining of trachea, muscles and other body wall tissues.
We use this protocol to examine microtubule organization in the developing sensor...

<table border="1">
	<tbody>
		<tr>
			<th>
				Name of the reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue 
				number</th>
			<th>
				Comments 
				(optional)</th>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>11251-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>FST</td>
			<td>15000-08</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse anti-&alpha;-tubulin (Clone: DM1A)</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td>Dilution 1/1000</td>
		</tr>
		<tr>
			<td>Mouse anti-Futsch (Clone: 22C10), 
			supernatant</td>
			<td>Developmental 
			Studies 
			Hybridoma Bank</td>
			<td>22C10</td>
			<td>Dilution 1/1000</td>
		</tr>
		<tr>
			<td>Rat anti-CD8 (Clone: 5H10)</td>
			<td>Caltag</td>
			<td>MCD0800</td>
			<td>Dilution 1/1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11001</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Cy3 anti-Rat IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>712-166-150</td>
			<td>Dilution 1/200</td>
		</tr>
	</tbody>
</table>

immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Watch this Scientific Journal Video about Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall at JoVE.com

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

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