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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 transcriptome^2,3. 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 tissue^4,5,6, 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 improvements^7,8, but adaptation was needed when applied to the isolation of enteric ganglia^8,9. 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.

Protokół

All protocols described here have been approved by the Vanderbilt University Institutional Review Board (IRB).

1. Preparation Prior to Tissue Arrival

1. 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.
   ​Note: This protocol can be adapted for use with mice and other rodent models.
   1. Immediately upon resection of each intestinal segment, thoroughly flush the lumen with chilled PBS or tissue-storage medium to remove residual luminal material or feces.
   2. After flushing and discarding waste in an appropriate biohazard receptacle, submerge the tissue in a sufficient volume of tissue-storage medium (such as 3-5 times the volume of intestinal tissue) and store in individually-labeled, water-tight plastic containers, submerged in ice or refrigerated until use).
   3. Process the tissue within 18-26 hours from the time of collection to prevent substantial RNA degradation.
      Note: Depending on the cleanliness of the intestinal segment and storage, it may be possible to extend our suggested time limit.
2. Prepare all materials needed for human tissue collection in advance, as indicated in this protocol (refer to the Table of Materials for more-detailed product information). Ensure that all required personal protective equipment is worn at all times.
3. Prepare dry ice buckets along with a dry ice slurry as shown in Figure 1.
   1. Crush enough dry ice to fill 4 standard circular ice buckets and one rectangular ice bucket. One of the standard buckets should have small (11 x 21 cm) laboratory tissues placed at the surface of the dry ice. If possible, use new, unopened boxes of laboratory tissues.
   2. Make a dry ice slurry by powderizing 2 L of dry ice in a clean rectangular ice bucket.
   3. Add pre-chilled 2-MB up to the surface of the dry ice. Slightly mix and flatten the slurry using a pipette tip box cover to shape surface of the slurry into a channel surrounded by larger pieces of dry ice along the sides of the rectangular bucket.
   4. Place several thin blocks of dry ice along the edges of the ice bucket to encourage more rapid freezing. There should be room for ≥4 base molds to fit loosely in the dry ice slurry bucket at a given time.
      1. Optionally, stack the ice bucket within another rectangular ice bucket filled ¼ with dry ice. This positioning helps better insulate the dry ice slurry and prevents the need for frequent adjustments of dry ice and 2-MB during the procedure.
   5. Position the dry-ice buckets in an assembly line in the following order: 1) dry ice slurry bucket, 2) laboratory tissue-dry ice bucket, 3 & 4) normal dry ice buckets, 5) rectangular dry ice bucket (Figure 1A).

2. Preparation of Flash-Frozen Human Intestinal Tissue

Note: This entire process can take between 45-80 min per intestinal segment, depending on the intestinal tissue quality. We also recommend collecting quality control samples to assess RNA quality and histology prior to proceeding with LCM.

1. Fill a large tray with wet ice and place surgical cutting boards atop the dry ice.
2. In this setup, chill a 500-mL and 100-mL beakers for storing the tissue and trimmed segments in cold storage solution. Pre-chill base molds on the cutting boards so that they are ready to receive tissue.
3. Upon receiving fresh intestinal tissue, pour off the media in which the tissue is transported and retain it in the pre-chilled beakers. Leave some room in these beakers for the temporary storage of the intestinal tissue and trimmed segments, which will be prepared in subsequent steps.
4. Cut the intestinal segment to a length of approximately 20 cm, which provides ample tissue (40-60 cm²) to generate RNA for downstream applications and quality control samples. Depending on the tissue integrity, it may be feasible to accomplish RNA isolation for downstream processing with a much smaller length (i.e., 5 cm of intestine).
5. Trim away adipose and connective tissue from the intestine using surgical scissors. Residual adipose along the intestinal serosa compromises the ability to fully flatten tissue in subsequent steps. Carefully trim the tissue, so as to avoid nicking the serosa during removal of fat and connective tissue, which can structurally damage the myenteric plexus or make the exterior of the tissue flatten unevenly for sectioning.
6. Make a longitudinal incision along the entire length of the intestinal sample, preferably at the site of mesenteric attachment.
7. Dissect away and discard the tenia coli from the colon, so that it flattens more readily, upon being released from tension.
8. Splay the intestine mucosa-side down onto a chilled cutting board and cut 1.25-cm-wide strips from the full length of the tissue.
   Note: Intestinal tissue often expands after trimming, so this size should be adjusted accordingly.
9. Temporarily store the strips in chilled tissue-storage solution.
   NOTE: We use Belzer UW cold storage solution because it has a long shelf life, is relatively cost-effective and can be stored at room temperature until needed.
10. Remove a tissue strip from the cold storage solution and cut it into segments ~1.5-2 cm long.
11. Quickly blot the intestinal segments on a stack of laboratory wipes, to remove excess liquid and prevent the formation of ice crystals along the intestinal serosa during flash-freezing. If tissue is not sufficiently dried, it will be difficult to obtain high-quality sections from the myenteric plexus.
12. Lay the blotted intestinal pieces mucosa-side up onto a pre-chilled, large, disposable plastic base mold (37 mm x 24 mm x 5 mm).
13. Carefully flatten each segment by lightly stretching the tissue over the surface of the base mold and using curved forceps to gently press down and expel any air bubbles that may be trapped beneath the serosa. Note that the tissue expands while flattening. If needed, trim the segments and cut the remaining samples at a smaller size.
    Note: If the tissue is over-stretched, this will distort the myenteric plexus. After all air bubbles are removed, assess the thickness of the sample prior to freezing. If needed, push the edges of the specimen inward until the intestinal sample approaches its original thickness.
14. Place the loaded base mold onto the surface of the dry ice, 2-MB slurry. The tissue should completely freeze within 30-60 s, depending on the size and thickness of the intestinal segment.
15. Dry the bottom of the base mold to remove residual 2-MB by quickly rubbing the base mold on the laboratory tissues pre-chilled in the second bucket of dry ice.
16. Transfer the dried sample to the third tub of dry ice and bury it under the surface of the dry ice until it is ready to be wrapped.
17. Quickly observe the bottom of the base mold prior to wrapping, to examine the quality of sample preparation. Make note of any samples that do not appear flat or have large air bubbles, as these should be avoided for cryosectioning and LCM.
18. Wrap the sample in pre-labeled aluminum foil that is laid atop dry ice in a bucket so that wrapping of tissue occurs while being kept completely frozen.
19. Wrap the sample with a layer of plastic wrap, to minimize tissue dehydration during storage at -80 °C.
20. Store samples in the rectangular bucket until they are ready to be moved to the freezer.
21. Pre-label and pre-chill freezer boxes at -80 °C for immediate storage of samples after collection.
22. Take a small portion of tissue from each intestinal segment for assessment of RNA integrity prior to conducting LCM.
    1. Pre-label a 2-mL RNase-free tube for each segment, filled with ≥500 µL of lysis buffer. We recommend using RNA Extraction Kit "D", listed in the Table of Materials.
    2. Homogenize a small (2 mm x 2 mm) piece of the intestine in a standard tissue micro-homogenizer (20-40 s, until no tissue chunks remain). Then, immediately freeze the RNA lysate on dry ice and store at -80 °C until proceeding with RNA extraction.
    3. Optionally, embed some of the sections in tissue freezing medium (TFM) as a back-up. Prepare tissue sections in the exact same way described in this section, but submerge samples in TFM within the base mold prior to flash-freezing.

3. Cryosectioning

1. Before cryosectioning, prepare all required materials for staining and dehydration and transfer the desired tissue samples from the -80 °C freezer into a bucket of dry ice.
2. Prepare the cryostat for use by setting to the optimal cutting temperature (-18 to -22 °C) and wiping down surfaces with 100% ethanol and treating the blade and brush tray with RNase decontamination solution.
3. To best adhere the sample to the chuck, use the following approach.
   1. Place forceps in dry ice for at least 30 s before unwrapping the intestinal sample and placing it at the surface of the dry ice serosa-side-up.
   2. Pour a 3-5 mm mound of tissue freezing medium (TFM) onto a large cryostat specimen holder and immediately transfer the intestinal sample serosa-side-up onto the TFM.
   3. Quickly transfer the specimen holder to dry ice and cover with powderized dry ice after allowing the TFM to adhere to the sample for 2-5 s at room temperature. Ensure that the TFM remains below the plane of the myenteric plexus.
      ​NOTE: Ideally, the outer surface of intestinal mucosa will fully adhere to the TFM before the TFM begins to freeze without thawing of the sample.
4. Mount the specimen holder into the cryostat's specimen head and align the specimen with the cryostat blade, such that the plane of the myenteric plexus will be parallel to the cutting blade.
5. Set the sectioning thickness on the cryostat to 8 µm. The purpose of perfectly aligning the specimen is to obtain the greatest possible surface area of myenteric plexus on each slide. This thickness is generally recommended for both human and rodent tissue, but can be adjusted, as needed.
6. Section through the serosa and longitudinal muscle until reaching the myenteric plexus.
   1. To locate the myenteric plexus, mount a sample section onto a slide and stain the section with an aqueous dye, such as 1% Cresyl violet or toluidine blue, etc.
   2. View the section under a light microscope at 40-2000X magnification to identify the junction between the longitudinal and circular muscle layers. Microscope parameters are provided in the Table of Materials. 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. A sheet of the outer longitudinal muscle layer then begins. Once the border between the longitudinal and circular muscle layers is reached, a portion of the enteric ganglia will be observed.
      NOTE: In the next several sections, the myenteric plexus will become very evident, with large swaths of ganglia being present within a single section (Figure 2C). If the tissue is not completely flat at the start of sectioning, then only a portion of the ganglia will be present in each section at a given time. Sometimes, the intestinal sample may have an inherently uneven myenteric plexus, despite the tissue appearing perfectly flat. This is especially true for duodenum samples. In our experience, these samples can take much longer to process with LCM, but sufficient RNA can be collected within a single day's session. The exact location of the myenteric plexus can vary considerably between samples, based on the tissue and its preparation prior to freezing.
7. Begin collecting serial sections for LCM, with optimal collections providing the greatest number of neurons and glia per slide. Depending on the surface area of the sample, multiple sections can be combined onto a single PEN-membrane slide.
8. Mount the sections onto PEN membrane slides, chilled briefly in the cryostat for 5-10 s. When mounting, the tissue should melt only slightly, maximally preserving RNA integrity.

4. Staining

Note: For an overview of the staining process, refer to Table 1. All solutions used are prepared in standard 50-mL conical vials. Treating the outside of the tubes with RNase decontamination solution does not appear to improve results (data not shown).

1. Prepare a saturated solution of 4% Cresyl violet in 95% ethanol at least one day in advance and fully re-suspend the Cresyl violet prior to loading the syringe.
   ​Note: The concentration of ethanol may need to be lowered for effective staining, as described in the discussion section. We have not tested whether using 50% or 75% ethanol during staining significantly reduces RNA integrity. Cresyl violet is light-sensitive and thus should be protected from light.
2. After mounting sections of myenteric plexus onto the slide, immediately submerge the slide in a conical vial of 95% ethanol (pre-chilled at -20 °C) for 30 s.
   1. If the sections did not fully adhere to the slide in the previous step, slightly warm the bottom of the slide with a gloved hand (a laboratory wipe should be placed in-between the slide and glove), for full adherence before submerging in 95% ethanol. This solution can be re-used for at least 3-6 slides without reducing RNA integrity.
3. Lay the slide face-up on a lab wipe placed atop a pre-treated, RNase-free container⁸.
4. Pre-fill a 3-mL syringe with 4% Cresyl violet and attach a 0.2-µm syringe filter.
   Note: We recommend cellulose acetate filters.
5. Apply 6-10 drops of stain directly onto the tissue sections⁸ and incubate for 10-30 s, or until sufficient dye is retained when de-stained. While staining, gently rotate the slide container by hand to ensure that the tissue sections are evenly coated with stain.
6. Pour off the stain and immediately de-stain the slide in a vial of 75% ethanol. Repeatedly submerge the slide until the excess dye has mostly seeped off of the sample. This may take between 10-20 s.
7. Finalize de-staining by repeatedly dipping the sample in a vial of 95% ethanol for 10 s. Submerge the sample repeatedly until all excess stain has been removed.
   1. Modify the destaining duration, as needed, to optimize the staining of ganglia. If too much stain is removed, the sample becomes too transparent and it may be difficult to visualize
      NOTE: This staining protocol has been optimized based on several publications^5,8,10,11 which required modification before satisfactory results were obtained. Therefore, the concentration of ethanol used in the dye and during the destaining process should be modified, when necessary, to obtain an optimal result.

5. Dehydration

1. Immediately dip the slide in 100% ethanol 2-3 times, for a total of 10-20 s.
2. Submerge the slide in anhydrous 100% ethanol for at least 30 s. As anhydrous ethanol is very hygroscopic, add molecular sieves (8-12 mesh) to the vial of 100% ethanol prior to the start of the procedure to remove any absorbed water and thus more completely dehydrate the sample⁵.
3. Repeatedly dip the slide in xylene for 15-20 s. This step fully removes the layer of ethanol on the slide. Add molecular sieves ahead of time to the tubes of xylene, to fully adsorb excess ethanol and water molecules⁵.
   CAUTION: Xylenes are classified as an irritant to the eyes and upper respiratory tract and should be used in a chemical fume hood.
4. Submerge the slide in xylene (with molecular sieves, 8-12 mesh) for at least 10 min.
   Note: A brief break can be taken after this step, if needed. Samples can be left in xylene for 4-5 hours without detrimental effects to staining, LCM or RNA yield/integrity.
5. Optionally, individually prepare several additional slides at this point. If preparing a second round of slides after completing LCM on all prepared slides, the tissue in the cryostat may need to be refaced, due to dehydration of the tissue surface. One to four sections may need to be discarded before usable sections can be collected.

6. Laser Capture Microdissection (LCM)

1. Remove the slide from xylene and air-dry it in a chemical fume hood for at least 1 min. Insert a cartridge of LCM caps into the LCM microscope stage. Load the slide onto the stage and acquire an overview image of the slide.
2. Identify the desired location for LCM and load a cap onto the slide.
3. Align the infrared (IR) laser and adjust its power and duration to make a 20-30 µm diameter capture spot.
4. Locate the ultraviolet (UV) laser and set an appropriate cutting speed and intensity.
5. Outline the desired ganglia to be collected using the LCM software, making sure to stay within the boundary of the collectable area on the cap (depicted in the slide overview of the software program as a green circle).
6. In each of the traces, adjust the IR spots such that there is at least one IR spot every 100-500 µm.
7. Press the IR/UV cut button to proceed with the collection of all marked ganglia.
8. Once collections are completed, either move the cap to a new location and repeat the collection process or examine the LCM cap at the QC station once the cap is sufficiently filled with ganglia (or until the recommended time limit of 60-80 minutes is reached).
9. If debris are present on the cap, wipe it away using a fine-tipped paintbrush that has been pre-treated with RNase decontamination solution, rinsed with nuclease-free water, and completely dried.
10. Carefully examine the cap by eye or with magnification under a bright field microscope to ensure all debris has been removed. If the debris cannot be removed through use of the pre-treated paintbrush, then use a fine pipette tip (RNase-free) to scrape away the debris.
11. Secure the cap onto a 0.5 mL microfuge tube filled with 230 µL of RNA lysis buffer (Buffer RLT from RNA Extraction Kit "B", with β-mercaptoethanol added at a concentration of 10 µL/mL).
12. Invert the cap, briefly vortex, and incubate at room temperature for 30 min.
13. Centrifuge at ≥ 5,000 x g for 5 min and then transfer the microfuge tube to dry ice.
    Note: The protocol can be paused here. RNA lysates can be stored at -80 °C without a substantial effect on RNA degradation for at least 1 week. If RNA isolation is performed the same day, then chill the samples on ice until they are ready for extraction.
14. Perform all additional collections within the recommended maximal time limit of 80-90 min after removing the slide from xylene. As the dehydrated sections are exposed to ambient humidity, RNases can gradually become reactivated. Restricting the collection period can minimize such effects and maximally preserve RNA integrity.

7. RNA isolation

1. Prepare an RNase-free workstation for RNA extractions.
2. Prepare all tubes and reagents according to the manual for the RNA Extraction Kit.
   Note: While there are many kits available for RNA isolation following LCM, we have had best success using the RNA Extraction Kit "B", listed in the Materials list. See Figure 5 for a side-by-side comparison of RNA extraction reagents.
3. Remove samples from the -80 °C freezer and rapidly thaw in a 37 °C water bath.
4. Immediately vortex thawed samples for 2-3 s to evenly distribute the guanidinium thiocyanate salts.
5. Combine each sample with an equal volume of 70% ethanol. Pool 2 or more lysates together, if necessary, to reach a sufficient RNA concentration.
6. Proceed as per the manufacturer's instructions in the manual for the RNA extraction process, using the protocol optimized for microdissected samples.
7. Elute RNA at the final step into the minimum recommended volume of nuclease-free water (14 µL).
8. Following RNA isolation, aliquot 1-2 µL of RNA from each sample into a new pre-labeled RNase-free tube for quality assessment/quality control with a microfluidics device that can visualize small quantities of RNA, such as a Bioanalyzer. Aliquot an additional 1 µL into a separate tube for quantification with a high-sensitivity RNA quantification kit.
   1. Adjust these steps, as needed, to obtain a sufficient amount of RNA for downstream analysis (i.e., pool a larger number of LCM caps prior to RNA extraction).
9. Store RNA at -80 °C until collecting enough samples for downstream analysis.

Wyniki

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...

Dyskusje

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...

Materiały

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