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 the larvae
<ol>
 <li> Collect 30-40 age-matched third instar larvae and wash them in ddH2O followed by brief rinses in RNAse Away (Sigma-Aldrich), and a final wash in ddH2O to remove RNAse Away. Wick off the excess ddH2O completely with a clean Kimwipe prior to embedding the larvae in OCT.</li>
 <li> Take a clean tissue embedding mold and layer it with a thin (1.5-2mm) layer of OCT, just enough to cover a single layer of larvae.</li>
 <li> Prior to embedding the larvae, if needed, cool the OCT to 0&deg;C in the tissue embedding molds. Do this by placing the mold containing OCT on a block of ice. This will help reduce larval movement during the embedding process.</li>
 <li> Place the clean larvae on the pre-cooled OCT and arrange them in parallel to one another. Once the larvae have been arranged, slowly fill the mold with OCT without disturbing the larval arrangement. Snap freeze the mold by placing it on a dry ice block. [Critical Step: Snap-freeze the larvae instantly to reduce movement.] This method will allow the larvae to be in a single plane, maximizing the number of cells available per section for capture.</li>
</ol>
If it is necessary to preserve the tissue morphology for identifying the cells of interest, or if the expected number of cells per section is found to be satisfactory then proceed directly to Step 11.
Alternatively if the cells are labeled specifically and can be identified with an easily identifiable marker such as GFP or RFP, but the number of cells per section is found to be low, then Steps 5-10 may be helpful for increasing the number of cells available for capture per section. These are optional steps and should be considered only if required, as a method for increasing the number of da neurons available for LCM per section when the other method fails to provide favorable results. [Caution: This method may disrupt the overall tissue morphology and make tissue identification based on specific spatial location very difficult.]
<ol start="5">
 <li> Wash 50-70 third instar larvae as described in Step 1. Place the larvae in a 1.5 ml microfuge tube containing 500 &mu;l of RNAse free 1X PBS.</li>
 <li> Dounce the larvae using a polypropylene pestle (USA Scientific), with 6-7 slow powerful strokes.</li>
 <li> Centrifuge the solution at 16,000 (x) g for 5-10 seconds (until the larval cuticles settle down to the bottom of the microfuge tube]. Discard the supernatant. The pellet should primarily consist of larval cuticle to which the PNS, including da neurons, is tightly adhered.</li>
 <li> Wash the cuticle pellet 2-3 times in 1X PBS and resuspend the pellet in 500 &mu;l 1X PBS. Spin the solution at 16,000 (x) g for 1 minute to form a compact pellet. Aspirate the supernatant completely using a fine Pasteur pipette.</li>
 <li> Carefully remove the pellet from the microfuge tube using a clean spatula, and wick off the excess PBS using a clean Kimwipe tissue. [Note: It is important to keep the pellet as compact as possible to increase the cell yield]. Place the compact cuticle pellet on a plastic mold containing a thin layer of OCT (1mm approx).</li>
 <li> Gently spread out the pellet in a thin circular area. Fill the mold with OCT, and freeze the tissue as described in Step 4.</li>
 <li> Using a cryostat, cut frozen sections at 5-8 &mu;m thickness on plain, labeled, uncharged, RNAse free glass microscope slides. [Critical Step: Position the tissue sections near the center of the slide.] </li>
 <li> Store the slides either directly on dry ice or at -80&deg;C in a clean slide-box until ready for microdissection. Perform LCM preferably within a week after sectioning the tissue.</li>
</ol>
PAUSE POINT
2. Dehydration and Cuticle Removal from Frozen Larval Sections
[Critical Step: All solutions for dehydration must be prepared fresh before each LCM session. Complete dehydration of frozen larval tissue sections is essential for achieving optimal microdissection efficiency]
<ol>
 <li> Remove the slides containing the frozen larval tissue sections from the -80&deg;C freezer and place them on dry ice.</li>
 <li> Remove a single slide from dry ice and immediately place it directly into a 50 ml conical tube filled with 70% ethanol fixative, followed by a short rinse in RNAse free ddH2O according to the recommended times presented in Table 1.</li>
 <li> The presence of larval cuticle in the frozen sections may impair the capture efficiency by preventing efficient LCM cap-cell contact which may lead to non-specific capture. If necessary, conduct the following steps (4-5) to clear the cuticle from tissue sections.</li>
 <li> Gently pipette 50 &mu;l of 2.5% trypsin directly onto the tissue sections and incubate for 5-30 seconds at room temperature. [Critical Step: This step may require optimization. The incubation time depends upon the tissue to be microdissected and section thickness. Longer incubation may clear everything including the cells of interest, whereas a short incubation may be insufficient to remove the cuticle]</li>
 <li> Rinse the sections briefly in a 50 ml conical tube containing RNAse-free ddH2O to remove trypsin, and loosely adhered cuticle fragments.</li>
 <li> Dip the slides sequentially in each of the remaining ethanol and xylene solutions for the recommended times (Table 1) to complete the dehydration process.</li>
 <li> Following the dehydration gradient, the slides are briefly dried under a mild air stream for 60-120 seconds at room temperature prior to performing LCM. [Caution: Dry the xylene completely from the tissue sections prior to performing LCM. Xylene is known to dissolve the LCM cap polymer resulting in microdissection failure].</li>
</ol>
<table border="1">
<tbody>
 <tr>
 <td>70% Ethanol (Fixative)</td>
 <td>3-10 Seconds</td>
 </tr>
 <tr>
 <td>ddH2O</td>
 <td>5-10 Seconds</td>
 </tr>
 <tr>
 <td>2.5% Trypsin Incubation</td>
 <td>5-30 Seconds</td>
 </tr>
 <tr>
 <td>ddH2O</td>
 <td>5-10 Seconds</td>
 </tr>
 <tr>
 <td>70% Ethanol</td>
 <td>60 Seconds</td>
 </tr>
 <tr>
 <td>95% Ethanol</td>
 <td>60 Seconds</td>
 </tr>
 <tr>
 <td>100% Ethanol</td>
 <td>120 Seconds</td>
 </tr>
 <tr>
 <td>100% Ethanol</td>
 <td>120 Seconds</td>
 </tr>
 <tr>
 <td>100% Xylene</td>
 <td>120 Seconds</td>
 </tr>
 <tr>
 <td>100% Xylene</td>
 <td>120 Seconds</td>
 </tr>
 <tr>
 <td>Air dry in a mild air stream</td>
 <td>60-120 Seconds</td>
 </tr>
</tbody>
</table> 
Table 1: Recommended procedure for LCM dehydration of frozen larval tissue sections.
3. Laser Capture Microdissection
PixCell IIe LCM instrument equipped with Fluor 300 epifluorescence optics optimized for EGFP was used for performing the LCM.
[Critical Step: If possible, perform the microdissection in a humidity-controlled room to prevent any reduction in microdissection efficiency due to increased humidity. Room humidity is a critical factor affecting the microdissection efficiency. Low room humidity can cause an increased static-cling resulting in non-specific capture, while high room humidity can result in low microdissection efficiency. We achieved optimal LCM efficiency between 25-50% relative humidity conditions.]
<ol>
 <li> Turn on the power for the microscope and laser control box.</li>
 <li> Load the CapSure cap holder assembly with HS LCM caps (Molecular Devices): Two cartridges of LCM caps may be loaded at one time.</li>
 <li> Open the Molecular Devices software and enter the experiment details including slide number and Cap lot number.</li>
 <li> Load a new HS LCM cap from the cartridge onto the PixCell IIe LCM instrument and position it correctly with respect to the joystick to ensure proper positioning of the cap in relation to the capture area.</li>
 <li> Place the microscope slide containing freshly dehydrated larval tissue sections on the microscope stage for microdissection.</li>
 <li> Locate the fluorescently labeled cells using the oculars or the computer screen. Due to the high specificity of ppk-GAL4,UAS-mCD8::GFP transgenic reporter line, the class IV da neurons can be easily identified by GFP fluorescence.</li>
 <li> Subject the tissue to LCM using CapSure HS LCM caps [for a detailed set of instructions for setting the instrument, focusing the laser and performing LCM see reference22].</li>
 <li> Adjust the "Power" and "Duration" laser pulse parameters to achieve a precise melted polymer spot, whose size corresponds to the selected laser spot size. Adjust the settings to customize the size of melted polymer area. The following parameters were used for microdissecting single class IV da neurons: Spot Diameter 7.5 &mu;m, laser strength 30-50 mW, laser time and 2-4 ms and 1-2 shots per cell were used. [Critical Step: the laser parameters required for a proper spot may vary with tissue thickness and room humidity conditions. Adjust the "Power" and "Duration" settings to achieve the required melted polymer spot size for microdissecting single or multiple cells.]</li>
 <li> To maximize specificity, microdissect cells with minimal overlap and a strong fluorescence. Each cap is capable of capturing numerous cell bodies. [Critical Step: To avoid RNA degradation, limit the total analysis time including dehydration and microdissection to less than 45 minutes]</li>
 <li> Once all the cells of interest have been captured, lift the HS LCM cap with the microdissected cells and placed it on a clean slide to confirm the presence of the captured cells and visually inspect the cap for presence of unwanted cells or debris.</li>
</ol>
4. RNA isolation from LCM derived cells
<ol>
 <li> Attach the cap containing the microdissected cells to an ExtracSure (Molecular Devices) device. Add 12&mu;l of RNA extraction buffer (PicoPure, Molecular Devices) to the cap surface containing the cells and connect an inverted thin-walled reaction tube (GeneAmp, Applied Biosystems) to the ExtracSure device. Place the assembly on an alignment tray (Molecular Devices) and cover it with an incubation block (Molecular Devices) pre-heated to 42&deg;C inside an incubator.</li>
 <li> Following a 30 minute incubation, centrifuge the tubes containing the extracts at 800 (x) g for 2 minutes. Close the tubes and store the cell extracts at -80&deg;C until ready for RNA purification.</li>
</ol>
PAUSE POINT
<ol start="3">
 <li> Extract and column purify the RNA according to the PicoPure (Molecular Devices) RNA extraction kit instructions. DNAse treatment is optional, and can be performed on column during the RNA purification as necessary. If required, multiple LCM samples can be pooled together during purification in a single column to increase the final RNA yield and can be eluted in a small volume (11-30 &mu;l) of elution buffer (PicoPure, Molecular Devices) and stored at -80&deg;C until ready for use. If desired a 1 &mu;l aliquot may be used to assess total RNA quality on a Bioanalyzer 2100 (Agilent Technologies, Inc.).</li>
</ol>
Representative Results
Laser capture microdissection (LCM) was used to isolate single, class-specific or multiple Drosophila da neurons from third instar larvae (Figure 1). LCM allows for a high degree of precision and specificity in the isolation of Drosophila da neuron cell bodies (Figure 2a-c). Moreover, the total RNA purified from these isolated da neurons was found to be of excellent quality as indicated by the presence of sharp 5.8S, 18S and 28S ribosomal RNA peaks when analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) (Figure 2d).
To assess the neuronal-specific enrichment of our isolated cells we performed real-time quantitative reverse transcription PCR (qRT-PCR) using the neuronal gene-specific marker, elav. For the qRT-PCR analyses, we calculated the relative levels of elav expression in the LCM microdissected da neurons and normalized this expression to that of our endogenous control (rp49) and relative to total RNA isolated from whole larvae using the &Delta;&Delta;Ct method23. These analyses revealed over 2.5 fold enrichment in the relative levels of elav in LCM micro-dissected da neuron samples as compared to whole animals (Figure 3).
<img src="/files/ftp_upload/2016/2016fig1.jpg" alt="Figure 1" /> 
Figure 1: LCM of Drosophila peripheral neurons. (a) Age matched third instar larvae are selected, washed and (b) embedded in a cryomold with OCT and frozen at -80&deg;C, (c) 8&mu;m serial tissue sections are created using a standard cryostat and (d) placed uniformly on clean glass slides. The glass slides with the affixed tissue sections are stored at -80&deg;C prior to LCM processing. (e,f) Immediately preceding LCM, the tissue sections are thawed and briefly fixed in 70% ethanol followed by brief rinse in ddH2O. (g) The slides are briefly incubated with trypsin and rinsed with ddH2O to remove cuticles (black) from the larval sections. (h,i) tissue sections without larval cuticle are then further dehydrated in an ethanol gradient and finally cleared in xylene. (j) The LCM cap with a thermolabile polymer is placed on the tissue section and the laser is pulsed on the selected fluorescent cell body. The laser pulse melts the polymer and engulfs the selected cell body. The polymer cap, along with the captured cell body is lifted, and (k) RNA extraction buffer is added to the cell. The captured cell, along with the RNA extraction buffer is incubated at 42&deg;C for 30 minutes and can be either stored at -80&deg;C, or directly processed for RNA purification.
<img src="/files/ftp_upload/2016/2016fig2.jpg" alt="Figure 2" /> Figure 2: LCM facilitates high precision capture of da neurons. (a) Representative image of a dehydrated and trypsin treated 8 micron tissue section prior to performing LCM, showing two class-IV da neurons labeled with GFP by the ppk-GAL4,UASmCD8::GFP reporter strain. Note, one of the neurons is highlighted (arrowhead) for capture. (b) Cell body of the highlighted neuron (arrowhead) is cleanly micro-dissected with high specificity from the tissue section. (c) End-on view of a single class-IV da neuron captured on the LCM cap. (d) Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) electropherogram of total RNA isolated from LCM-derived da neurons, showing excellent RNA quality as indicated by the presence of sharp 5.8S, 18S, and 28S rRNA peaks. 
<img src="/files/ftp_upload/2016/2016fig3.jpg" alt="Figure 3" /> Figure 3: qRT-PCR reveals substantial enrichment of neuronal marker gene expression in LCM captured da neurons relative to whole animal. qRT-PCR analyses of neuron-specific marker gene expression (elav) in LCM captured da neurons (21-7-GAL4,UAS-mCD8::GFP) and whole animals in age matched third instar larvae was performed in triplicate. Relative levels of elav expression in LCM captured da neurons were normalized to the endogenous control (rp49) and relative to whole larvae using the &Delta;&Delta;Ct method23. A 2.5 fold enrichment in the relative levels of elav in LCM captured da neuron samples was observed relative to whole animals.
Troubleshooting
Problem: RNA degraded, or of low quality.
Ensure RNAse free condition throughout experiment. Try reducing amount of time spent on LCM per slide to 30 minutes. Keep the slides and samples on dry ice except when performing LCM. Avoid thawing the tissue sections once they are cut.
Problem: Most cells are lost from the slide during trypsin treatment (step 17-18).
Try reducing the time of trypsin treatment. Try reducing the trypsin concentration.
Problem: Presence of cuticle and other non-specific tissue debris on the cap polymer.
Try removing the tissue debris by blotting the polymer surface with the tacky side of an adhesive note22.
Problem: Low LCM efficiency.
Try increasing the incubation time in 100% ethanol and xylenes. Reduce room humidity using dehumidifiers.

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No conflicts of interest declared.

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>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<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

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13.4K Views.  George Mason University. 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.

Video: Laser Capture Microdissection of Drosophila Peripheral Neurons

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 development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

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

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron 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

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

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  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

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

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 is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

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 (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

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 between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

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

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 carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

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 sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

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

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.