Name: optimization of laser-capture microdissection for the isolation of enteric ganglia from fresh-frozen human tissue
Uploaded: 2018-06-14T14:30:00
Description: Watch this Scientific Journal Video about Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue at JoVE.com

We are grateful to the donors and their families who made this work possible. We also appreciate the assistance of staff at the International Institute of Medicine and Tennessee Donor Services for helping coordinate collection of tissue used in this study. This work was supported by grants from the National Institutes of Health, NIH OT2-OD023850 to EMS2 and stipend support from NIH T32-DK007673 to AMZ. We are grateful to staff of the Vanderbilt Translational Pathology Shared Resource for access to the LCM Instrument and advice on tissue preparation. The Vanderbilt Tissue Pathology Shared Resource is supported in part by NIH grants P30-CA068485-14 and U24-DK059637-13. We thank the Genome Technology Access Center in the Department of Genetics at Washington University School of Medicine for help with genomic analysis. The GTAC is partially supported by NCI Cancer Center Support Grant #P30 CA91842 to the Siteman Cancer Center and by ICTS/CTSA Grant #UL1TR002345 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. This publication is solely the responsibility of the authors and does not necessarily represent the official view of NCRR or NIH.

All protocols described here have been approved by the Vanderbilt University Institutional Review Board (IRB).
1. Preparation Prior to Tissue Arrival
<ol>
	<li>Obtain proper IRB approval and coordinate with human organ donation agencies to obtain freshly-resected, unfixed intestinal tissue from donors that meet all research criteria required for the study. 
	&#8203;Note: This protocol can be adapted for use with mice and other rodent models.
	<ol>
		<li>Immediately upon resection of eac...

<ol>
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This procedure enables the efficient collection of numerous enteric ganglia as a source to derive RNA for RNA-seq. Here, we have accelerated the processes outlined in existing protocols while maximally preserving RNA integrity. As all steps in this procedure are interdependent, it is important that all issues be eliminated from the onset of the study and that all samples are prepared as similarly to one another as possible, to obtain reliable RNA-seq.
From the onset of the procedure, RNA integ...

This method is designed to obtain high-quality RNA samples of enteric ganglia from human intestinal tissue using laser capture microdissection (LCM). The protocol described here has been optimized to provide sufficient RNA quality and yields for RNA sequencing (RNA-seq) and is intended to be used with freshly-resected, unfixed, flash-frozen human intestinal tissue.
Functional gastrointestinal and gut motility disorders affect one of every four people in the United States. The enteric nervous system (ENS), also referred to as the second brain1, is often at the center of these disorders, as it plays a crucial role in gut homeostasis and motility. Manipulation of gut motility has generally been restricted to surgical resection of the aganglionic/noncontractile tissue, chronic dietary modification and/or medications. Surprisingly, the full transcriptome of the adult ENS remains to be sequenced, greatly limiting our ability to identify molecules within the ENS that can be targeted pharmaceutically or utilized in stem cell therapies.
There are relatively few methods for isolating RNA from human enteric ganglia. The first approach, cell dissociation2, requires high incubation temperatures and long incubation times; both of which are known to promote RNA degradation and alter the transcriptome2,3. An alternative approach, LCM, more reliably preserves the transcriptome and protects RNA integrity. Although several studies have used LCM to collect ganglia from fresh-frozen human intestinal tissue4,5,6, these approaches were either hampered by poor RNA quality and quantity, were quite labor-intensive, or needed modification of staining or RNA extraction techniques to work in our hands. Other LCM protocols designed for preserving RNA that were found in LCM product manuals provided additional improvements7,8, but adaptation was needed when applied to the isolation of enteric ganglia8,9. For these reasons, we developed an optimized protocol based on these resources that yields substantial quantities of high-integrity RNA from human enteric ganglia, has a relatively fast workflow, and produces consistent results across a large number of samples.
In this study, we present a synopsis of optimized procedures that facilitate the isolation of high-integrity RNA from enteric ganglia sourced from resected human intestinal tissue. Our method incorporates five important aspects. First, freshly-resected, unfixed human intestinal samples should be trimmed to size, have all excess moisture removed with a laboratory tissue and flattened in a large base mold before flash-freezing atop a slurry of dry ice and 2-methylbutane (2-MB). Second, histologic sections of intestine should be prepared to obtain the full plane of the myenteric plexus on a slide, which offers a large payload of enteric ganglia. Success with this step is largely dependent on the tissue preparation process. Third, the nonuniform structure of ganglia in the ENS requires the use of polyethylene napthalate (PEN) membrane slides6, which offer the greatest speed and precision during the LCM process. Fourth, ethanol-compatible dyes, such as Cresyl Violet, should be used to preserve RNA integrity while staining enteric ganglia. Last, the RNA extraction process is critical for a successful outcome with RNA-Seq. We sought an RNA extraction approach that produces high RNA integrity, maximizes RNA yields when starting with small collections of enteric ganglia, eliminates DNA contamination, and retains as many RNA species as possible.
Taken together, optimization of these factors in the present study greatly accelerates the workflow and yields samples of enteric ganglia with exceptional RNA quantity and quality. Results have been largely consistent among a sizable group of samples, indicating the consistency of this approach. Further, we have used these approaches to successfully sequence dozens of RNA samples from enteric ganglia. The strategies highlighted here can also be broadly adapted for performing LCM of desired ganglia or nuclei of the peripheral and central nervous system and other cases requiring the isolation of high-quality RNA.

<table border="0" cellpadding="0" cellspacing="0" style="width:920px;" width="919">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">10% Neutral-buffered formalin</td>
			<td style="width:280px;">Sigma</td>
			<td style="width:131px;">HT501128-4L</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-Methylbutane</td>
			<td>Fisher</td>
			<td>O3551-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-mL syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-mL conical tubes, polypropylene&#160;</td>
			<td>Corning</td>
			<td>05-526B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Base molds 37x24x5mm, disposable</td>
			<td>Electron Microscopy Sciences</td>
			<td>5025511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Belzer UW&#160; Cold Storage Solution</td>
			<td>Bridge to Life</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CapSure Macro LCM caps</td>
			<td>Arcturus, ThermoFisher</td>
			<td>LCM0211</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cling Wrap, Press&#8217;n Seal&#160;&#160;</td>
			<td>&#160;Glad&#160;&#160;</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cresyl Violet Acetate</td>
			<td>Acros Organics</td>
			<td>AC405760025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cutting Board</td>
			<td>Electron Microscopy Sciences</td>
			<td>63308</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 190-proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000190</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 200-proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Microscope slides</td>
			<td>Fisher</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gloves, extended cuff</td>
			<td>Microflex&#160;</td>
			<td>19010144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gowns, surgical-disposable</td>
			<td>Kimberly-Clark&#160;</td>
			<td>19-088-2116</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tray, 20 L (large polypropylene sterilizing pan)</td>
			<td>Nalgene&#160;&#160;</td>
			<td>1335920B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kimwipes (large)</td>
			<td>Kimberly-Clark&#160;</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kimwipes (small)</td>
			<td>Kimberly-Clark&#160;</td>
			<td>34133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser Capture Microdissection System (ArcturusXT)</td>
			<td>ThermoFisher</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leica Cryostat Chuck, large,&#160; 40 mm&#160;</td>
			<td>Southeast Pathology Instrument Service</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Light Microscope</td>
			<td>Olympus</td>
			<td>CX43</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope objective (20X)</td>
			<td>Olympus UPlanFl 20X/0.50, &#8734;/0.17</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope objective (10X)</td>
			<td>Olympus UPlanFl 10X/0.25, &#8734;/-</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Sieves</td>
			<td>Acros Organics</td>
			<td>AC197255000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease-free water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PicoPure RNA extraction kit</td>
			<td>Applied Biosystems</td>
			<td>12204-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pellet Pestle</td>
			<td>Kimble Kontes</td>
			<td>4621973</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEN membrane LCM slides</td>
			<td>Arcturus, ThermoFisher</td>
			<td>LCM022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-free tubes, 1.5 mL</td>
			<td>Ambion</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNaseZAP&#160;</td>
			<td>Sigma</td>
			<td>Sigma-R2020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;A&#34; (PicoPure)</td>
			<td>Applied Biosystems</td>
			<td>12204-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;B&#34; (RNeasy&#160; Micro kit)</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Method &#34;C&#34; (TRIzol)</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;D&#34; (RNeasy PLUS Mini kit)</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Splash Shield, disposable faceshield</td>
			<td>Fisher</td>
			<td>17-310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors, Surgical, 14.5 cm &#34;sharp-sharp&#34;</td>
			<td>Fine Science Tools</td>
			<td>14002-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe filter, cellulose acetate (0.2 &#956;m)</td>
			<td>Nalgene&#160;&#160;</td>
			<td>190-2520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Freezing Medium</td>
			<td>General Data Healthcare</td>
			<td>TFM-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylenes</td>
			<td>Fisher</td>
			<td>X3P1GAL</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We have made several improvements to existing protocols that enable the relatively rapid collection of enteric ganglia from human intestinal samples using LCM, meeting the standards for RNA-Seq. First, we optimized the rapid freezing of intestinal tissue segments in large base molds placed at the surface of a slurry of dry ice in 2-MB (Figure 1B). The best success during cryosectioning and subsequent LCM was obtained by laying intestinal segm...

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

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 ...

This method can help answer key questions about the enteric nervous system, such as the expression profile of enteric ganglia in the intestines of adult humans. The main advantage of this technique is that it can be used to obtain high yields of RNA from human enteric ganglia while maximally preserving RNA quality. Begin with intestinal tissue cut into 20 centimeter lengths.First, trim away the adipose and connective tissue. Avoid nicking the serosa, which can lead to structural damage to the myenteric plexus. Next, make a longitudinal incision along the entire length of the trimmed sample, preferably at the site of mesenteric attachment.Then, dissect away and discard the taeniae coli from the colon so the tissue can flatten out. Now, splay the intestine, mucosa side down, onto a chilled cutting board. Then, cut 1.25 centimeter wide strips along the full length of the tissue.As they are cut, transfer each piece to chilled tissue storage solution for temporary storage. Next, cut each strip into segments that are two to 1.5 centimeters long. After slicing a strip into segments, blot the segments dry.It's important that the tissue is very dry so ice crystals don't form on the tissue when it is frozen. Now, arrange the dried pieces of tissue, mucosa side up, on a cold, disposable plastic mold. Then, flatten each piece using curved forceps to remove trapped air bubbles.The tissues will expand as they are flattened. After flattening the tissue segments, assess their thicknesses prior to freezing. If needed, push the edges of the specimen inward until the intestinal sample approaches its original thickness.Now, transfer the loaded base mold onto a slurry of dry ice and 2-methylbutane. The tissue should completely freeze within 30 to 60 seconds. Then, immediately dry the bottom of the mold using cold wipes that have been kept on dry ice.Now, check the sample. Take note of any samples that do not appear flat. Next, wrap the mold in aluminum foil that has been pre-labeled and chilled before use.Then, cover the foil with a layer of plastic wrap to minimize dehydration. Keep the samples on ice until they can be stored at minus 80 degrees Celsius. After preparing the required materials, pour a three to five millimeter mound of TFM onto a large cryostat specimen holder and immediately transfer the intestinal sample, serosa side up, onto the TFM.After a few seconds at room temperature, transfer the specimen holder to dry ice and cover it with powderized dry ice. Before slicing, make sure that the TFM is below the plane of the myenteric plexus. Next, mount the specimen holder into the cryostat specimen head and align the specimen with the cryostat blade.Make the plane of the myenteric plexus parallel to the cutting blade. Now, make eight micron sections through the serosa and longitudinal muscle until reaching the myenteric plexus. To locate the myenteric plexus, mount a sample and stain it with an aqueous dye, such as 1%crystal violet.To proceed, identify the junction between the longitudinal and circular muscle layers. At the start of sectioning, the serosa will be marked by the presence of connective tissue. Some remaining mesenteric fat may also be present along the serosa.Then, the sheet of the outer longitudinal muscle layer becomes visible Once the border between the longitudinal and circular muscle layers has been reached, a portion of the enteric ganglia will be observed. In the next several sections, the myenteric plexus will become very evident with large swaths of ganglia being present within a single section. Now, collect serial sections for the laser-capture microdissection.Chill PEN slides in the cryostat for five to 10 seconds and use them to mount the sections. The best sections will have a lot of neurons and glia. Now, proceed to stain and then dehydrate the sections as described in the text protocol.In preparation, insert a cartridge of LCM caps into the LCM microscope stage. Then, load the sample slide onto the stage and acquire an overview image of the slide. Identify the desired location for LCM and load a cap onto the slide.Then, align the infrared laser and adjust the laser's power and duration for a 20 to 30 micron diameter capture spot. Next, outline the desired ganglia to be collected. Make sure to stay within the boundary of the collectible area on the cap, which is displayed as a green circle.For each of the ganglia outlined by a UV cutting trace, adjust the IR spots such that there is at least one IR spot every 100 to 500 microns to ensure the ganglia will all be collected when the cap is lifted off of the sample. Then, press the IR UV Cut button to proceed with the collection of all the marked ganglia. Once collections are completed, move the cap to a new location and repeat the collection process.This can be repeated for 60 to 80 minutes. When the LCM cap is loaded with enough ganglia, transfer it to a quality control station. If debris is present on the cap, wipe it away using a fine-tipped paint brush that has been prepared for RNA use.Use a pipette tip for any difficult to remove debris. Then, secure the cap onto a 0.5 milliliter microfuge tube filled with 230 microliters of RNA lysis buffer. Invert the cap, briefly vortex it, and then incubate the sample at room temperature for 30 minutes.Later, centrifuge the sample for five minutes at 5, 000 g or faster, and then transfer the microfuge tube to dry ice. The sample is now ready for the RNA extraction. When pulling two caps of enteric ganglia per sample, greater than one nanogram of RNA was obtained, enough for RNA-Seq, and the RNA was of excellent quality.The RNA integrity of shown sample is 8.3. Samples with integrity numbers of 7.5 and 6.9 were also collected. In total, more than 95%of all prepared samples have been good enough for RNA-Seq.The quality of the RNA-Seq data sets was assessed using a bioinformatics pipeline. The end bias indicates that the samples were successfully sequenced and only had a modest three prime end bias. A gene biotype plot shows that the sequencing results were largely consistent.Once mastered, this technique can be used to collect biological replicates of RNA from enteric ganglia of sufficient quality and quantity for RNA-Seq in as little as eight to 12 hours. While attempting this procedure, it is important that the human intestinal samples be frozen as flat as possible. This will ensure that you can collect very large cross-sections of human enteric ganglia that are quickly and easily processed with laser-capture microdissection.

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Research

JoVE Journal

Neuroscience

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.

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.

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

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

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 ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

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 ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...