We thank Drs. Yuh-Nung Jan and Wes Grueber for providing fly stocks used in this study, and Virginia Espina, Dr. Emanuel Petricoin and Dr. Lance Liotta for assistance with LCM. The authors acknowledge the Thomas F. and Kate Miller Jeffress Memorial Trust for support of this research (D.N.C.) and the George Mason University Provost s Office (E.P.R.I.).

General Comments on LCM of Drosophila Peripheral Neurons
Allow from 6 hours, up to a week or longer for LCM depending upon the tissue type and the number of cells required.
All the procedures are carried out in strictly RNAse-free conditions following standard procedures. Larvae expressing either 21-7-GAL4,UAS-mCD8::GFP or ppk-GAL4,UAS-mCD8::GFP transgenic reporter lines were used for these experiments.
1. Preparing...

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The protocol presented herein describes our optimized method for the isolation of Drosophila peripheral neurons via LCM. While this LCM protocol was designed for the specific isolation of single, class-specific or multiple Drosophila da neurons from the third instar larval stage of development, minor modifications of the protocol could readily be adapted for the capture of other Drosophila cell types from all developmental stages using distinct GAL4,UAS-mCD8-GFP reporter transgenes wh...

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		<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>10X Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>MP Bioproducts</td>
<td>PBS10X02</td>
<td>Diluted to 1X working solution</td>
</tr>
<tr>
<td>2.5% Trypsin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1426</td>
<td>&nbsp;</td>
<tr>
<td>RNase-AWAY</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>83931</td>
<td>&nbsp;</td>
<tr>
<td>Xylenes, histological grade</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>X3S-4</td>
<td>&nbsp;</td>
<tr>
<td>RNAse-free water</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP561-1</td>
<td>&nbsp;</td>
<tr>
<td>Optimal Cutting Temperature (OCT) compound</td>
<td>&nbsp;</td>
<td>Tissue-Tek</td>
<td>4583</td>
<td>&nbsp;</td>
<tr>
<td>PicoPure RNA Isolation Kit</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>KIT0204</td>
<td>Follow manufacturer&#x2019;s instructions</td>
</tr>
<tr>
<td>Thin walled reaction tube with domed cap</td>
<td>&nbsp;</td>
<td>GeneAmp, Applied Biosystems</td>
<td>N8010611</td>
<td>&nbsp;</td>
<tr>
<td>ExtracSure Sample Extraction Device</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>LCM 0208</td>
<td>&nbsp;</td>
<tr>
<td>Incubation Block for sample extraction from CapSure HS Caps</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>LCM0505</td>
<td>&nbsp;</td>
<tr>
<td>Alignment Tray for CapSure HS LCM Caps and ExtracSure Sample Extraction Devices</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>LCM0504</td>
<td>&nbsp;</td>
<tr>
<td>CapSure HS LCM caps</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>LCM0213</td>
<td>&nbsp;</td>
<tr>
<td>75x100 mm glass slides</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-544-3</td>
<td>&nbsp;</td>
<tr>
<td>Tissue embedding molds	</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>NC9642669</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene pestle for 1.5 ml microcentrifuge tubes</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1415-5390</td>
<td>&nbsp;</td>
<tr>
<td>70% Ethanol; 95% Ethanol; 100% Ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dry ice</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			Equipment:
<ul>
<li>Cryostat</li>
<li>50 ml conical tube for slide fixation, rinsing, trypsin treatment and ethanol/xylene dehydration</li>
<li>-80&deg;C freezer</li>
<li>Incubator</li>
<li>PixCell IIe LCM Instrument with Fluor 300 epifluorescence optics optimized for EGFP (Molecular Devices-Molecular Devices)</li>
</ul>		</div>

laser capture microdissection of drosophila peripheral neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Watch this Scientific Journal Video about Laser Capture Microdissection of Drosophila Peripheral Neurons at JoVE.com

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

laser-capture-microdissection-of-drosophila-peripheral-neurons

Research

JoVE Journal

Neuroscience

13.3K vistas.  George Mason University. En este video-artículo se presenta un método para aislar uno o varios Drosophila Neuronas da a partir de larvas de tercer estadio con la captura de infrarrojos (IR) de la clase Laser Microdisección Captura (LCM). ARN obtenido de las neuronas aisladas puede ser fácilmente utilizada para aplicaciones posteriores como QRT-PCR o microarrays de análisis.