Name: laser capture microdissection of drosophila peripheral neurons
Uploaded: 2010-05-24T17:00:00
Description: Watch this Scientific Journal Video about Laser Capture Microdissection of Drosophila Peripheral Neurons at JoVE.com

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...

<ol>
	<li>Corty, M. M., Matthews, B. J., Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecules+and+mechanisms+of+dendrite+development+in+Drosophila.">Molecules and mechanisms of dendrite development in Drosophila.</a> Development. 136, 1049-1061 (2009).</li><li>Parrish, J. Z., Emoto, K., Kim, ., 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=Mechanisms+that+regulate+establishment,+maintenance,+and+remodeling+of+dendritic+fields.">Mechanisms that regulate establishment, maintenance, and remodeling of dendritic fields.</a> Annu. Rev. Neurosci. 30, 399-423 (2007).</li><li>Wang, X., Starz-Gaiano, M., Bridges, T., Montell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+specific+cell+populations+from+Drosophila+tissues+by+magnetic+bead+sorting,+for+use+in+gene+expression+profiling.">Purification of specific cell populations from Drosophila tissues by magnetic bead sorting, for use in gene expression profiling.</a> Nature Protocols. , (2008).</li><li>Iyer, E. P. R., Iyer, S. C., Sulkowski, M. J., Cox, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+Drosophila+peripheral+neurons+by+magnetic+bead+sorting.">Isolation and purification of Drosophila peripheral neurons by magnetic bead sorting.</a> J Vis Exp. 34, 2912-2917 (2004).</li><li>Tirouvanziam, R., Davidson, C. J., Lipsick, J. S., Herzenberg, L. A., Tirouvanziam, R., Tirouvanziam, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+cell+sorting+(FACS)+of+Drosophila+hemocytes+reveals+important+functional+similarities+to+mammalian+leukocytes.">Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes.</a> Proc. Natl. Acad. Sci. 101, 2912-2917 (2004).</li><li>Shigenobu, S., Arita, K., Kitadate, Y., Noda, C., Kobayashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+germline+cells+from+Drosophila+embryos+by+flow+cytometry.">Isolation of germline cells from Drosophila embryos by flow cytometry.</a> Dev. Growth Differ. 48, 49-57 (2006).</li><li>Reeves, N., Posakony, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+programs+activated+by+proneural+proteins+in+the+developing+Drosophila+PNS.">Genetic programs activated by proneural proteins in the developing Drosophila PNS.</a> Dev. Cell. 8, 413-425 (2005).</li><li>Jinushi-Nakao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knot/Collier+and+Cut+control+different+aspects+of+dendrite+cytoskeleton+and+synergize+to+define+final+arbor+shape.">Knot/Collier and Cut control different aspects of dendrite cytoskeleton and synergize to define final arbor shape.</a> Neuron. 56, 963-978 (2007).</li><li>Yang, Z., Edenberg, H. J., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mRNA+from+specific+tissues+of+Drosophila+by+mRNA+tagging.">Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.</a> Nucl. Acids Res. 33, (2005).</li><li>Appay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+Gene+Expression+Profiling+of+Human+T+Cell+Subsets+Reveals+Parallel+Post-Thymic+Differentiation+for+CD4++and+CD8++Lineages.">Sensitive Gene Expression Profiling of Human T Cell Subsets Reveals Parallel Post-Thymic Differentiation for CD4+ and CD8+ Lineages.</a> J. Immunol. 179, 7406-7414 (2007).</li><li>Ginzinger, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+quantification+using+real-time+quantitative+PCR:+An+emerging+technology+hits+the+mainstream.">Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream.</a> Exp. Hematol. 30, 503-512 (2002).</li><li>Keays, K. M., Owens, G. P., Ritchie, A. M., Gilden, D. H., Burgoon, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+and+single-cell+RT-PCR+without+RNA+purification.">Laser capture microdissection and single-cell RT-PCR without RNA purification.</a> J. Immunol. Methods. 302, 90-98 (2005).</li><li>Volgin, D. V., Swan, J., Kubin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RT-PCR+gene+expression+profiling+of+acutely+dissociated+and+immunocytochemically+identified+central+neurons.">Single-cell RT-PCR gene expression profiling of acutely dissociated and immunocytochemically identified central neurons.</a> J. Neurosci. Methods. 136, 229-236 (2004).</li><li>Emmert-Buck, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection.">Laser capture microdissection.</a> Science. 274, 998-1001 (1996).</li><li>Xiao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+in+embryonic+mouse+lenses.">Gene expression profiling in embryonic mouse lenses.</a> Mol. Vis. 12, 1692-1698 (2006).</li><li>Scharschmidt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+human+osteoarthritic+connective+tissue+by+laser+capture+microdissection+and+QRT-PCR.">Analysis of human osteoarthritic connective tissue by laser capture microdissection and QRT-PCR.</a> Connect. Tissue Res. 48, 316-323 (2007).</li><li>Spletter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=regulates+Drosophila+olfactory+projection+neuron+identity+and+targeting+specificity.">regulates Drosophila olfactory projection neuron identity and targeting specificity.</a> Neural Dev. 2, 14-14 (2007).</li><li>Hoopfer, E. D., Penton, A., Watts, R. J., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+Drosophila+neuronal+remodeling:+a+role+for+the+RNA-binding+protein+Boule+as+a+negative+regulator+of+axon+pruning.">Genomic analysis of Drosophila neuronal remodeling: a role for the RNA-binding protein Boule as a negative regulator of axon pruning.</a> J. Neurosci. 28, 6092-6103 (2008).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22, 451-461 (1999).</li><li>Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Song, W., Onishi, M., 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=Peripheral+multidendritic+sensory+neurons+are+necessary+for+rhythmic+locomotion+behavior+in+Drosophila+larvae.">Peripheral multidendritic sensory neurons are necessary for rhythmic locomotion behavior in Drosophila larvae.</a> Proc. Natl. Acad. Sci. USA. 104, 5199-5204 (2007).</li><li>Espina, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection.">Laser capture microdissection.</a> Nat. Prot. 1, 586-603 (2006).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.</a> Methods. 25, 402-408 (2001).</li></ol>

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...

<!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>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 ...

Research

JoVE Journal

Neuroscience

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...