Method Article

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

DOI:

10.3791/60503

⸱

December 18th, 2019

 ,  ,  ,  , 
1Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 2Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai

In This Article

Summary

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This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Abstract

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Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel’s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

Introduction

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The musculoskeletal system is a multicomponent system composed of muscle, connective tissue, tendon, ligament, cartilage, and bone, innervated by nerves and vascularized by blood vessels1. The skeletal tissues develop with increasing cellular heterogeneity and structural complexity. Cartilage and bone develop from the same osteochondroprogenitor lineage and are highly related. Embryonic cartilage and bone develop in association with muscles, nerves, blood vessels, and undifferentiated mesenchyme. Cartilage may also be surrounded by bone, such as Meckel's cartilage and condylar cartilage within the mandibular bone. These tissues are anatomically associated and interact with each other through extracellular signals during development. In the study of gene expression in the development of cartilage and bone, one obstacle is the heterogeneity of skeletal structures composed of multiple tissue types. Precise isolation of the specific tissue of interest is key for successful transcriptional analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate cell types or regions of interest within heterogeneous tissues, and is reproducible and is sensitive to the single cell level2. It can precisely target and capture cells of interest for a wide range of downstream assays in transcriptomics, genomics, and proteomics3,4. The quality of the isolated RNA, DNA, or protein can be assessed with a bioanalyzer or equivalent platform. For example, RNA quality is indicated by the RNA integrity number (RIN)5.

Here, we provide a protocol for the rapid staining and isolation of cartilage and bone by LCM from fresh frozen tissues. We use the mouse embryo to demonstrate that this protocol yields high quality RNA for subsequent transcriptomic analysis, such as RNA sequencing (RNA-seq).

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Protocol

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Tissues from mice were obtained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and study protocols were approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai.

1. Preparation of Fresh Frozen Specimen

  1. Dissect the embryo or tissue of interest. Embed the sample in a disposable embedding mold with optimal cutting temperature (OCT) compound. Adjust the orientation of the specimen with a tip or needle.
  2. Rapidly freeze the samples in a dry ice/methyl-2-butane bath. Continue to the next step or store at -80 °C.
    NOTE: The protocol can be paused here.

2. Cryosectioning for Laser Capture Microdissection

  1. Defrost the cryostat and clean internal surfaces with 70% ethanol. Treat the anti-roll plate and a pair of forceps with RNase decontamination agent. Set the cryostat to the desired cutting temperature (-18 to -22 °C). Prepare a lidded container with dry ice, placing a sheet of aluminum foil on the dry ice for temporary storage of the sectioned sample slides during cryosectioning.
    CAUTION: Dry ice is extremely cold. Always handle dry ice with care and wear insulated gloves whenever handling it.
  2. Transfer the fresh frozen specimen into a bucket of dry ice. Place a layer of OCT compound onto a cryostat specimen holder and immediately place the specimen on top of the OCT with gentle pressure. When OCT is completely frozen, fasten the holder with the specimen into the cryostat cutting arm.
  3. Leave the sample in the cryostat for 15 min to equilibrate to the cutting temperature.
  4. Section the tissue and collect the slices on polyethylene naphthalate (PEN) membrane slides at a thickness of 12 μm. Align consecutive sections on the membrane. Once one slide is completed, allow sections to dry for a few minutes and transfer the slide onto the foil in the dry ice container until all the required slides are collected.
  5. Store the slides at -80 °C in a slide box.
    NOTE:Section thickness is chosen to maximize the amount of tissue collected while allowing efficient cutting of the tissue, and may vary depending on tissue of interest. The protocol can be paused here. Keep slides free from RNase contamination. Avoid temperature changes. Although the RINs of RNA from slides stored for up to 6 months may still indicate high quality of the RNA, we recommend using slides as soon as possible.

3. Sample Preparation for Laser Capture Microdissection

  1. Prepare solutions for staining and dehydration.
    1. Place 45 mL of 80% ethanol in each of three 50 mL centrifuge tubes on ice. Label them as #1, #2, and #3. Dilute ethanol with RNase/DNase free water.
    2. Place 45 mL of 95% ethanol in a 50 mL centrifuge tube on ice.
    3. Place 45 mL of 100% ethanol in a 50 mL centrifuge tube on ice.
    4. Place 45 mL of xylene in a 50 mL centrifuge tube at room temperature.
      CAUTION: Xylene should be used in a fume hood.
  2. Thaw a PEN membrane slide briefly by placing it against a gloved hand, then wash twice for 30 s each in 45 mL of 80% ethanol (#1 and #2) with agitation in 50 mL centrifuge tubes to remove the OCT.
    NOTE: It is important to remove OCT before staining, to prevent OCT loosened during staining from obscuring the sections. Forceps can be used to facilitate removal of OCT that is not washed off by agitation.
  3. Lay slide on a sheet of aluminum foil. Pipette 0.8 mL of 0.1% cresyl violet in 50% ethanol onto the slide and stain for 30 s. Wash the slide in 45 mL of 80% ethanol (#3) for 30 s, and then dehydrate by passage for 30 s each through 45 mL of 95% ethanol, 100% ethanol, and xylene in 50 mL centrifuge tubes.
  4. Stand slides on a delicate task wiper for 5 min to drain xylene and dry sections.
    NOTE: Slides must be dried completely before LCM.

4. Laser Capture Microdissection

NOTE: Wear powder free nitrile gloves while performing LCM.

  1. Turn on the LCM microscope, laser, and computer. Log on to computer and start the software. Turn the key to the "On" position to start up the laser. When the green light (Laser) is on, press the red button to activate the laser, and then the light (Laser) turns red indicating laser is ready to use.
    NOTE: The Laser needs approximately 10 min to warm up.
  2. Set up the collection tubes.
    1. Insert the cap of a 0.5 mL polymerase chain reaction (PCR) tube into the collector.
      NOTE: Choose the matching collector for the desired PCR tubes (0.2 or 0.5 mL).
    2. Pipette 50 µL of extraction buffer (provided by RNA isolation kit listed in the Table of Materials) into the cap of the 0.5 mL collection tube.
    3. Insert the collection device into the microscope.
    4. In the Change Collector Device window, click on the Move to Reference Point button to move the collector to the reference point (RP). Adjust the focus to clearly view the RP. Click the arrows to move the RP to the center of the view.
  3. Load specimen slides.
    1. Click the left Unload button in the toolbar to lower the slide holder.
    2. Place the slide into the holder, with the tissue section facing downwards.
      NOTE: This orientation ensures that the captured tissue sections fall into the cap of the collection tube by gravity.
    3. Insert the holder back into the stage.
  4. Set up the laser parameters.
    1. Select the Control option of the Laser menu or click on the Laser icon.
    2. Adjust these parameters as desired: Power, the power of the laser; Aperture, the width of the laser; and Speed, the speed of cutting in Draw and Cut mode.
      NOTE: Test the laser in an area out of interest. Higher power or larger aperture makes it easier to cut but causes more damage to cells. Adjust the combination of power, aperture, and speed to obtain efficient cutting and minimal damage of the tissue. For example, Power = 40, Aperture = 5, and Speed = 7 for cartilage tissue on a 12 μm section.
  5. Select and cut areas of interest.
    1. Start with 5x objective and find the areas of interest, and then switch to the objective (5x, 10x, or 40x) that best shows the area of interest.
      NOTE: After cresyl violet staining, cartilages are stained magenta and mineralized tissues appear brown or black, distinguishable from other tissues. Images can be saved using Save Image As from the File menu.
    2. Choose the tube for collection (A, B, C, D) by clicking the mark at the collector.
    3. Choose Move and Cut or Draw and Cut. In Move and Cut mode, the specimen is cut manually, using the mouse or the touch-screen pen to draw shapes freehand. In Draw and Cut mode, shapes may be drawn freehand with the mouse or touch-screen pen for subsequent cutting. Click the Start Cut button to initiate laser cutting.
    4. Once the cut is completed, the target tissue with PEN membrane falls into the cap of the collection tube by gravity. Repeat 4.5 to pool multiple areas of interest if needed.
      NOTE: Repeating cuts or increasing Power or Aperture may be necessary if the tissue does not drop into the cap of the collection tube due to incomplete cutting. Mineralized bone (brown or black) cannot be cut directly by laser.
  6. Unload the collector and carefully close the PCR tube. Place the microdissected tissues in dry ice. Continue to the next step or store at -80 °C.
    NOTE: The protocol can be paused here. Repeat 4.2 to 4.6 for the next sample.

5. Lysis of Microdissected Tissues and RNA Isolation

  1. Thaw the microdissected tissues at room temperature. Centrifuge briefly and incubate the samples for 30 min at 42 °C.
  2. After incubation, spin the lysis buffer down into the 0.5 mL PCR tube. Perform DNase treatment and RNA extraction using an RNA isolation kit (see the Table of Materials) following the manufacturer's instructions.

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Results

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Coronal sections of fresh frozen mouse tissues at E16.5 were used to demonstrate the isolation and collection of Meckel's cartilage (MC), condylar cartilage, and mandibular bone by LCM. Mouse embryos at E16.5 were dissected and embedded in cryogenic molds with OCT compound. Samples in molds were rapidly frozen in a dry ice and methyl-2-butane bath and stored at -80 °C.

To demonstrate cresyl violet staining of cartilage and bone, cryosectioning in the coronal plane was performed and samples wer...

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Discussion

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LCM enables the isolation of enriched or homogenous cell populations from heterogeneous tissues. Its advantages include rapid and precise capture of cells in their in vivo context, while potential disadvantages include it being time consuming, expensive, and limited by the need for the user to recognize distinct subpopulations within a specified sample30. This protocol provides details of LCM of mouse embryonic cartilage and bone, highlighting the use of cresyl violet staining in a rapid ...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was supported by the National Institute of Dental and Craniofacial Research (R01DE022988) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01HD078233). The authors thank the Biorepository and Pathology Core for access to the Leica LMD 6500 platform at the Icahn School of Medicine at Mount Sinai.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
2-MethylbutaneThermoFisher ScientificO3551-4
BioanalyzerAgilentG2939BA
Centrifuge tubeThermoFisher Scientific339653Conical sterile polypropylene centrifuge tubes, 50 mL
Cresyl violet acetateSigma-AldrichC5042
CryostatLeica BiosystemsCM3050 S
Delicate task wiperThermoFisher Scientific06-666
Disposable embedding moldThermoFisher Scientific1220
Distilled waterInvitrogen10977-015DNase/RNase-Free
Ethanol, absolute (200 proof)ThermoFisher ScientificBP2818Molecular biology grade
Glass PEN membrane slideLeica Microsystems11505158
LCM systemLeica MicrosystemsLeica LMD6500
Microscope cover glassThermoFisher Scientific12-545FP
Microscope slidesThermoFisher Scientific12-550-15
OCT compoundElectron Microscopy Sciences102094-106
PCR tube with flat cap, 0.5 mLAxygenPCR-05-CLCM collection tubes
Permanent mounting mediumVector LaboratoriesH-5000
RNA isolation kitThermoFisher ScientificKIT0204
RNase decontamination agentSigma-AldrichR2020RNase decontamination agent for cleaning surfaces
XyleneSigma-Aldrich214736

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Tags

Bone Tissue IsolationCresyl Violet StainingRNA Integrity PreservationCryosectioning TechniqueTissue Dehydration ProtocolMeckel s CartilageMandibular Bone

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