Name: optimization of laser-capture microdissection for the isolation of enteric ganglia from fresh-frozen human tissue
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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.

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The authors have no conflicts of interest to disclose.

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 integrity can be negatively impacted if the time interval (cold ischemic time) between collection of tissue at the time of organ donation and receipt of tissue for processing in the laboratory is too long. Given this concern, we were surprised that high-quality RNA can still be extracted from intestinal samples even after 24 hours of cold ischemic time. When the intestinal samples are chilled and stored properly in cold storage solution, there is superb protection of tissue integrity and RNA quality. We require that the intestinal tissue is flushed upon collection by the organ collection team, to limit the exposure of the intestine to digestive enzymes, RNases and other molecules that could impact RNA quality during this period. We have found that when the intestinal specimen is not completely flushed, this can significantly reduce the sample&#39;s RIN.
Using our optimized procedure for preparing intestinal tissue samples, we are able to avoid embedding samples in TFM. Because TFM interacts with ethanol and xylene to form a dark brown precipitate that interferes with the visualization of enteric ganglia, we prefer excluding it from our samples. However, when working with intestinal tissue from mice or other species, the exclusion of TFM may not be possible. In this case, we have experimented with removing TFM from an adhered section on a slide. If the tissue has a sufficient surface area, ethanol (chilled at -20 &#176;C) can be dripped onto the slide with a transfer pipette to quickly fix the TFM, allowing it to be removed with forceps. Dripping ethanol onto the slides avoids the accumulation of TFM within the vial of ethanol, which can exacerbate the browning effect when multiple slides are being processed.
During flash-freezing, we favor using a dry ice slurry with 2-MB, rather than 100% ethanol, because 2-MB readily evaporates when landing on the surface of the tissue. Ethanol tends to evaporate more slowly, especially when combined with TFM, forming a chemical sludge on the tissue block that cannot be easily removed. Both ethanol and 2-MB tend to fizz and splatter vigorously when freezing in small freezing containers, because the small volume of the dry ice slurry has a low specific heat capacity. Instead, using a large rectangular bucket filled with powderized dry ice provides a large cooling mass and prevents temperature fluctuations while the tissue is being frozen.
As mentioned, it is sometimes easier to obtain continuous sheets of enteric ganglia from some tissue samples relative to others. Understandably, the overall speed of LCM is subject to variability depending on the characteristics of the source tissue or the intestinal region being sampled. For this reason, it is often best to prepare many fresh-frozen samples at the onset. We have generally found that a total of 20 samples per segment of intestine (~2 cm x 1.5 cm, each) is far more than sufficient to provide ample RNA for downstream applications. However, if only a small length of intestinal tissue is available, we recommend a minimum length of 5 cm. With our approach, the lowest yield that would be obtained would range from 18-30 ng of RNA, as further discussed below. The minimum 5 cm length can potentially be reduced, depending on the application, especially if higher amounts of cDNA preamplification are used. These parameters were not tested in the current study. For example, enteric ganglia have been collected from full-thickness intestinal biopsy samples (2 cm x 2 cm) for use with qPCR4.
As we demonstrate in this study, the use of the ethanol-compatible dye, Cresyl violet, offers much better protection of RNA integrity than the aqueous stain, toluidine blue. When submerged in ethanol, RNases become inactive5,15. However, RNases can still regain function once exposed to water7,15. When staining with aqueous dyes, the tissue must be exposed to nuclease-free water for nearly 1 min9, which leads to substantial RNA degradation. When using toluidine blue tissue staining, we were unable to reduce the exposure to water before and after staining, because such modifications also reduced the uptake and retention of toluidine blue, respectively. A similar problem was also encountered with a hematoxylin-based dye9. In the modified protocol described here, staining with Cresyl violet avoids direct exposure of the intestinal samples to water, thereby maximally preserving RNA integrity. These results eliminate the need for additional RNase inhibitors in the staining solution. We found that such inhibitors are ineffective at enabling the use of the aqueous dye, toluidine blue, as it interfered with both the uptake and retention of the dye.
When initially optimizing our staining protocol, we were unable to reproduce the results obtained in other protocols and reports5,7,8,11. While the reason for this is unclear, we found several factors that led to inconsistent dye-uptake and retention. For example, although certain protocols have successfully stained tissue using Cresyl violet in 100% ethanol8 or in 75% ethanol5, the dye only faintly labeled ganglia or had high background, compared to using Cresyl violet in 95% ethanol. Last, the percentage of Cresyl violet used in the staining solution is also very important. Most protocols recommend a 1% Cresyl violet solution7,8, but this concentration did not provide reliable staining in our hands, even after staining sections for up to 2 min. We had best success staining with a clarified saturated staining solution of 4% Cresyl violet11 applied to the sample through a sterile syringe filter8. This saturated solution enables much more rapid staining of the sample, in as little as 10-15 s. Pre-fixation of the sample in chilled 95% ethanol (at -20 &#176;C) enables a more rapid uptake of the stain.
Another report found that enteric ganglia were most distinctly stained in fresh-frozen human intestinal sections when combining Cresyl violet with eosin in a 75% ethanol solution5. However, we found that eosin made it more difficult to identify ganglia and that Cresyl violet, alone, offers superior detection. In a different study, it was found that a buffered Cresyl violet solution provided consistent staining results for endometrial cancer tissue10. However, we found this did not offer any improvement for human intestinal tissue and also could not be implemented when using 95% ethanol for the staining solution.
We have tested a number of conditions in which the protocol can be paused and resumed at a later time. Many protocols suggest sectioning slides ahead of time and re-freezing them at -80 &#176;C immediately after mounting onto the slide5,9. With this approach, staining and LCM can then be resumed within the next week, offering great flexibility. However, not only did this greatly interfere with staining; it also reduced RNA integrity in our hands (data not shown). To circumvent this issue, Grover et al. recommend staining and dehydrating the sections prior to storage in the freezer11. In this approach, slides are stained and dehydrated (as in Table 1) up to the 100% anhydrous ethanol step (protocol section 5.2). Then, samples are incubated in a second vial of 100% anhydrous ethanol for 10 min and are transferred immediately to a conical tube with desiccant gel, which is then chilled on dry ice and transferred to the -80 &#176;C freezer. When removing samples from the freezer, slides are immediately submerged in 100% anhydrous ethanol for 30 s and air-dried prior to performing LCM11. Unfortunately, in our hands, this approach reduced RIN by 0.6-0.8 (data not shown). We therefore resorted to performing all sectioning, staining, and LCM in the same day in order to obtain maximal RNA integrity in our conditions. This proves to be a time-consuming process, but 10-14 LCM caps can be filled during a full day&#39;s work.
The most reliable point at which our protocol can be paused is at the xylene incubation step. Slides have been left in xylene (with molecular sieves) for up to 4-5 h without any apparent effect on RNA integrity. We have found that when three slides are prepared at a time, all can be processed with LCM within this timeframe, even if an hour break is needed. An alternative option is to desiccate slides after staining and dehydration steps within a vacuum-sealed exicator16, but we have not yet attempted this approach.
RNA isolation is yet another crucial step to the procedure, with the most important factors being: obtaining the maximal possible yield of RNA, retaining the full spectrum of RNA (without loss of smaller RNA species)17,18, and completely eliminating genomic DNA from the sample17. In our hands, RNA Extraction Kit &#34;B&#34; provided the best outcome with our samples, as it offered greater yields and was more efficient at genomic DNA elimination. Our observations regarding differences in yields between RNA extraction reagents are consistent with prior reports17,18. However, there are also many LCM studies that have reported success with other RNA extraction reagents19,20. Prior reports also indicate that the column-based RNA extraction processes offer better retention of small RNA molecules than the phenol-based RNA Extraction methods18 as small RNA molecules can be lost in the supernatant during RNA precipitation steps.
We observed that RNA lysis buffers that use guanidinium thiocyanate with added &#946;-mercaptoethanol is highly effective at removing all captured ganglia from the LCM cap (Figure 5D). While most LCM protocols do not suggest vortexing the lysate, we have found that this produces excellent results and does not result in RNA degradation. It also provides an even greater likelihood that all captured ganglia will be released from the cap for RNA extraction. Using these methods, we generally obtain RNA yields from LCM of enteric ganglia in the range of 1.8 to 18 ng/cm2 of intestinal tissue, depending on how many ganglia are present on a given slide. Our RNA-seq procedure requires a minimum input of 10 ng RNA, but other procedures can have much lower RNA input if a sequencing processing includes a greater number of pre-amplification cycles. The total surface area of tissue stored from the preparation and flash-freezing process is 40-60 cm2, which provides ample RNA for cDNA library prep and low-input RNA-Seq applications.
While our procedure is designed for the collection of whole enteric ganglia, the approach can be readily modified for the collection of single neurons, or glia, if desired5. However, because glial processes densely ensheath neurons of the ENS, glial RNA will be included along with the RNA of captured neurons, albeit at lower counts. This raises issues when trying to identify neuronal transcripts expressed at a low copy number. Single-cell RNA-seq offers the best precision and detection in this regard, but requires neurons to be dissociated from the intestinal tissue, stained with pan-neuronal markers, and isolated with flow-cytometry21 or identified from whole-intestinal dissociations after sorting with bioinformatics approaches. The downside of most dissociation studies is that most protocols require that the sample be incubated at room temperature or 37 &#176;C for extended periods, which is known to alter the transcriptome3. Recently, the cold-active protease from Bacillus licheniformis has been used for generation of single cell suspensions for mouse tissues and it may be possible to apply this enzyme for dissociation of human tissue at 4 &#176;C22. Until such optimization is accomplished, LCM of fresh frozen human tissue is a robust means to capture RNA for expression profiling of peripheral nervous system elements like enteric ganglia.
In summary, we have developed a reliable and consistent protocol for the collection of RNA from human enteric ganglia. We have optimized the preparation of human intestinal tissue, staining, LCM process, and RNA extraction based on numerous publications and protocols. We have also demonstrated the reliability of this approach in collecting RNA samples of sufficient RNA quality for RNA-Seq. This protocol can be applied broadly to tissues collected from patients with a number of gastrointestinal diseases and can be readily adapted for use with rodent models, thereby offering a reliable strategy for the development of novel therapeutics.

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

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

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 ...

optimization-laser-capture-microdissection-for-isolation-enteric

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.6K Views.  Vanderbilt University Medical Center. Het doel van dit protocol is het verkrijgen van hoge-integriteit RNA monsters van darmen ganglia geïsoleerd van nietgefixeerde, vers gereseceerd menselijke intestinale weefsel met behulp van de laser vangt microdissection (LCM). Dit protocol omvat voorbereiding van monsters van menselijke intestinale weefsels, cryosectioning, ethanolbevattend kleuring en uitdroging, LCM, flash-bevroren en RNA extractie.

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

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 ...

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 ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Medicine

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 ...

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.

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

Biology

Laser Microdissection: A Technique to Isolate Invading Colorectal Cancer cells from a 3D Organotypic Culture Model

Source: Bullock M. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/51475"> Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and&#160;Ex vivo&#160;Fibroblasts.</a> J. Vis. Exp. (2014).
The video describes the laser dissection of the 3D organotypic co-culture model with micron-level precision. In 3D organotypic models, stromal constituents such as fibroblasts are 3-dimensionally co-cultured with cancer epithelial cells. The 3D model serves as an experimental tool that enables invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Source: Bullock M. et al.,  Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and&#160;Ex ...

JoVE Encyclopedia of Experiments

Cancer Research

Colorectal Cancer

Assays & techniques

Cryopreserved Tissue Section Fixation and Staining: A Procedure to Process Frozen Brain Tissue Sections for Laser Microdissection

Submerge the slides in 30-second sequential descending ethanol immersions, followed by crystal violet solution staining for 20 seconds and 5 seconds in 0.5% eosin Y solution.
At the end of the eosin Y incubation, use filter paper to blot the slides dry and dehydrate the slides in sequential ascending ethanol immersions. After the 60-second ethanol immersion, rinse the slides in a container of xylene for 3 minutes.
Next, dry the slides on RNase-free surface at room temperature for 10 seconds before mounting the samples in mounting medium prepared with RNase-free water. After 10 to 20 seconds, transfer the slides onto the microscope microdissection platform.

Cancers of the Nervous System

In vitro studies

EoE Sub Articles

Isolation of Cells from Sensory Ganglia

After harvesting the sensory ganglia, aspirate 500 microliters of ganglia dissociation solution from each tube without disturbing the ganglia. Next, add 1 milliliter of PBS to each sample and wait for the ganglia to settle to the bottom of the tubes.
Then, remove the PBS and add 1 milliliter of ganglia dissociation solution supplemented with digestion enzyme to the neurons. Incubate the tubes at 37 degrees Celsius for the appropriate time, based on the age of the digestion enzyme, shaking and flicking the tubes briefly every 15 minutes to ensure the ganglia are covered by the digestion solution.
While the nerve cells are being digested, slowly and carefully add 400 microliters of freshly prepared 12% density solution over 400 microliters of 28% density solution in one 1.5-milliliter tube per sample. Two distinct layers should be visible, one darker than the other.
At the end of the digestion period, discard the digestion enzyme, and wash the ganglia two times with 1 milliliter of PBS as just demonstrated. After the second wash, add 200 microliters of fresh ganglia dissociation solution to each tube.
Then, using a 200-microliter pipette set to a 100-microliter volume, pipette the ganglia up and down several times to dissociate the cells into a single-cell suspension, taking care to avoid bubbles. When no intact tissue pieces are visible, holding a 70-micron cell strainer firmly in one hand, pipette the dissociated cell solution into the center of the strainer. Then, wash the dissociation tube with 100 microliters of fresh ganglia dissociation solution to collect any additional cells.
Filter the wash through the strainer, and use a new pipette tip to aspirate any remaining cell-containing solution on the underside of the strainer, adding it to the cell suspension. Now, carefully layer all 300 microliters of the cells on top of the previously prepared density gradient, and collect the ganglia cells by centrifugation.
At the end of the centrifugation, carefully remove the top 700 microliters of solution containing the majority of the cell debris. Then, mix 700 microliters of fresh ganglia dissociation solution with the remaining cells. Pellet the cells by centrifugation, and carefully discard the supernatant.

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

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