Expression Microdissection Adapted to Commercial Laser Dissection

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© 2011 Nature America, Inc. All rights reserved. Tissues processed with various fixatives were analyzed in the the in analyzed were fixatives various with processed Tissues NIH the at in-house modified were EVAthat clear films using source, laser 400-mW fixed a and stage motorized a instru with ment prototype a as well as dissections, for microscope laser analysis. molecular procures section cells histological immunostained for subsequent the from film the of Removal cells. target to it bonding and film dissection the of activation heat focal chromogen,causing the by ates the entire tissue section ( ically (IHC) stained cells and exposed to a light source that irradi film is applied to a histological slide containing immunohistochem molecular targeting using cells of on operator-independent based selection dissection laser for improvements substantive offers that advance practical platforms. analysis downstream some for biomolecules of amounts large relatively for requirement the and precision improved for need the as such clinic-based researchers. However, this field has certain challenges, and laboratoryof ies are by used now a routinely spectrum wide advanced the molecular pathology field, and microdissection stud and proteomes transcriptomes genomes, cellular of analysis in-depth and quantitative facilitating improved, have protein and RNA DNA, analysis image and software platforms computerized of the and incorporation instruments dissection available commercially of several development the with substantially, evolved has microdissection technology laser 1990s, the in invention original its Since Development of the protocol I molecular analysis. to complete the protocol is highly dependent on the target cell population and the number of cells needed for subsequent Moreover, xMD can provide improved dissection precision because of the unique characteristics of film activation. many applications by using a targeting probe for cell procurement in place of an operator-based, cell-by-cell selection process. order to make the technique widely available to the biomedical research community. adaptation of xMD to commonly used laser microdissection instruments and to a commercially available handheld laser device in in dissection however,capabilities; until recently the method has not been accessible to investigators. for molecular analysis. L Published online 17 March 2011; doi:10.1038/nprot.2010.202 the work. Correspondence should be addressed to M.R.E.-B. ([email protected]). Biophysics, National Institute Child of Health and Human Development, National Institutes Health,of Bethesda, Maryland, USA. 3 2 1 Jaime Rodriguez-Canales Jeffrey C Hanson laser dissection instruments Expression microdissection adapted to commercial samples; initial study, including formalin-fixed, paraffin-embedded (FFPE) Signal Signal Processing and Section, Instrumentation Center for Information Technology, National Institutes Health,of Bethesda, Maryland, USA. Pathogenetics Unit, Pathology,of Laboratory Center for Cancer Research, National Cancer Institute, National Institutes Health,of Bethesda, Maryland, USA. Laser Microdissection Core, Pathology,of Laboratory Center for Cancer Research, National Cancer Institute, National Institutes Health,of Bethesda, Maryland, USA. NTRO aser-based aser-based microdissection facilitates the isolation of specific cell populations from clinical or animal model tissue specimens Our original proof-of-concept xMD publication used a PixCell II Expression microdissection (xMD) represents a conceptual and D

UCT 1–4 ethanol-fixed, paraffin-embedded (EFPE) specimens; and . In parallel, protocols for analyzing small amounts of of amounts small analyzing for protocols parallel, . In 5–10 I ON . Current laser-based instruments have successfully have successfully instruments laser-based . Current 1 1 1 . For xMD, a clear ethylene vinyl acetate (EVA) , 5 , Michael A Tangrea E xpression microdissection (xMD) is a second-generation technology that offers considerable advantages Fig. 1 1 & Michael R Emmert-Buck a ). The light energy is absorbed 2 , 5 , Skye Kim 2 , Michael D Armani 1 1 , 1 2 - - - - .

and cancerous tissue cancerous and against CD31 and Factor VIII to analyze endothelial cells in normal a third study used a similar xMD strategy and targeting antibodies AE1/AE3-positive cells for subsequent epigenetic analysis, and then xMD films to dissect smooth muscle actin-positive and cytokeratin In study, second a xMD in-house system and II PixCell a we used 3-phosphate dehydrogenase, E-cadherin and smooth muscle actin. AE3, CD3, prostate-specific antigen, desmin, S-100, glyceraldehyde proliferating cell nuclear antigen, cytomegalovirus, cytokeratin AE1/cryopreserved tissue. Targeting probes included antibodies against visualization of cells of interest. In this approach, IHC or or IHC approach, this In interest. of cells of visualization Fendby developed process itself upstream tissue processingthe of effects and not to the dissection to related is biomolecules recovered the of quality the and cells the to damage no or little is thus,there tissue; the by not and dye or chromogen the by absorbed mostly is energy IR LCM,ard the appropriate buffer for molecular an into analysis. placed are they For and both cells xMD bonded and the procures stand tissue the to targets the bond and melt partially to it causing polymer the in dye the by absorbed is energy light The operator’sguidance. the under cells IR laser is activated under microscopic and aimed at visualization EVA is polymer applied onto thermoplastic the slide ( scope micro a through cells target of identification visual on based is As opposed to xMD, standard laser capture microdissection (LCM) other methods with Comparison research community. laser sources to make xMD technology accessible to the biomedical studies,these previous but it has been adapted to several different Immuno-based laser dissection is a follow-on technique technique follow-on a is dissection laser Immuno-based 1,2 . A histological section is stained, and then a dye-containing 1,2,14–16 1,2,14 2 , Thomas J Pohida . Subsequent removal of the dissection film from.film dissection the Subsequent removal of T . he he method improves dissection speed for et al. et 12,13 . The protocol presented here is based on based is here presented protocol. The natureprotocols 1 7 that uses immunotargeting to improveto immunotargeting uses that 5 These These authors contributed equally to T 3 his his protocol describes the , Robert F Bonner

4 | Laboratory of Medicalof Laboratory VOL.6 NO.4VOL.6 T he he time protocol Fig. 1 | 2011

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© 2011 Nature America, Inc. All rights reserved. lenging and tedious process that, even when successful, dissectionsrecoversusingstandard lasermicroscopes extremelyanis chal only anoperator-independent manner. In contrast, performing precise tion, all targeted cells or targeted organelles are rapidly procured in nuclei ing a higher resolution microdissection such as endothelial cells or area activationof and subsequent capture are reduced, thus allow the size of the targeting laser beam in standard laser dissection), the generated by the adjacent chromogen on target cells (as opposed to tion of EVA is strictly a consequence of theof the uniquefocal physical temperatureproperties of film-cell interaction.increase As activa precision and throughput. analyses: dissection-based facilitate that xMD of advantages tant than competitive with, existing approaches. There are two impor to,rather complementary be will technique the that and inquiry will extend microdissection capability into new areas of biological defined cells in sections.histological Our is expectation that xMD dissection tool for investigators studying phenotype or molecularly films such as polyethylene naphthalate membrane slides of types specialized of use the for allow protocols IHC improved research neuroscience and studies environment micro tumor analysis, epigenetic including articles, published tion. has been used for Immuno-based laser numerousdissection by a human operator is typically needed to ensure proper cell selec recognition software; however, visual identification of positive cells automatic via cells target immunolabeled of dissection facilitated has microscopes laser into programs analysis image of poration incor recently,the Moremanner. one-by-one a in visualization cific cells that the laser operator then procures under microscopic immunofluorescence is used as a visualization tool to identify spe is manually fired at the targets in a one-by-one manner to generate a heat transient that melts EVA and ofbonds standardit to thelaser underlyingcapture microdissection. cells. First, under direct microscopic visualization, the operator identifies target cells in theunderlying tissue section.target Then, cells.the Nolaser microscopic visualization or operator-based cell selection is required during the xMD process. ( chromogen melts the EVA polymer and bonds it to increases focally at the site at which the section is irradiated. The transient temperature covered with an EVA film and the entire tissue Target cells are selectively immunolabeled, ( Figure 1 458 a protocol Box 1 or kitdepends onthe biomolecule(s) of interest and the method of tissuepreservation. Standard buffersand kitsusedfor laser-microdissected samplescanbeusedtoextract captured tissuefrom xMDfilms. The exact buffer ) Schematic showing the principle of xMD. The precision of microdissection using xMD is improved because The adaptation of xMD to available instruments provides a new

• • • • • •

| VOL.6 NO.4VOL.6 (55 °C) and agitation. Do not heat the film in the SDS buffer above 80 °C because the film will melt and compromise sample recovery.For denatured protein analysis (1D-PAGE), we use the Novex Tris-glycine SDS sample buffer (2×; Life Technologies) with gentle heating orbital heater. sufficient extraction buffertocoverthe filmwhile incubating the tubewith regularagitation using anEppendorf themomixer oran Following xMD,the ethylene vinyl acetate film withtransferred cellsshould beplacedinto a1.5-mlEppendorf tubecontaining For protein mass spectrometry, weuseaprotocol described byJohann after xMDisbetteraccomplished using mass spectrometry. proteins from immunostained tissuesections varies depending onthe tissue typeand staining method. Ingeneral, protein profiling For mildprotein analysis (2D-PAGE), weusethe Total Protein Isolation Kit(ITSI Biosciences); however, note thatthe recovery of For RNAextraction, weusethe PicoPure kit(LifeTechnologies). For DNA,weusethe QIAampDNAMicro kit(Qiagen). 11,13

| Schematic diagrams of xMD and LCM. . Importantly, because xMD is based on a probe for dissec | E | 2011 X TRACTI |

natureprotocols O N FR O M x a Activated polymer MD FILMS 17–25 Moreover, . 2 2 . Glass slide ------transcriptome amplification methods that can be used to analyze used be can that methods amplification transcriptome of availability the by investigators for mitigated is difficulty this Box 1 per to (see techniques dissection immuno-based the challenging of any using is form and procedure immunostaining the by compromised is sections frozenmRNAtissue in of measurement to the study of mRNA and microRNA in archival samples; however, xMD can be successfully applied to DNA analysis, proteomics and steps, followed by enzymatic-based colorimetric labeling. Currently, The immunostaining process includes several incubation and wash protocols the general rule of thumb in the dissectionanalysis field is is one ‘thesuch moreexample increase in the number of dissected cells is advantageous; proteomic able to amplification methods. For many assays, though,small amountsa significant of input material or are nucleic-acid LCMbased work andwell amenfor downstream molecular assays that either require generated from precisely dissected cells or organelles. 10 organelles in a histological section, whether it contains 10 targets or assays. Because xMD can procure the probe-targeted all of cells or a few targets and scant biomolecules with limited use in molecular that IHC staining has on the biomolecules in tissue sections tissue in biomolecules the on has staining IHC that staining instrument that rapidly xMD,processes slides. of capability especially high-throughput when performed in the concert with an automated of immuno advantage by improvedtaking be may below) see cryosections, from mRNA for be measured. Thus, even some low-abundantcan moleculesandmoderate- more asincreased is amount of starting material increases, and the depth of the analysis cellsthe better’, as the molecular data are typically improved as the With respect to throughput, standard laser dissection and immuno- However, caveat effect is the for technology xMD an important 6 targets, relatively large amounts of DNA, RNA or protein can be et al. ) 24,25 9 . Although this is a limitation of the current xMD method, . Tissue section Chromogen Clear EVApolymer IR laser 4 . Moreover, even for b Activated polymer

amplification-based studies (except b ) Schematic showing the principle Glass slide

amplification-based Tissue section Dyed polymer Plastic cap IR laser

24,25 - .

© 2011 Nature America, Inc. All rights reserved. we accomplished this by using an air is essential for xMD. For the original prototype ing, as good contact between the EVA polymer and the ciency,tissue section we tested variations in film-tissue pressure and film preheat EVA the of Preparation polymer. body concentration used for IHC should be titrated accordingly. activation that requires strong staining; therefore, the primary anti willgenerally notbe procured. There distinctisa threshold film of weakly stained cells or any cells or intensityorganelles is that stronglywith staineda cells ‘blushwill be efficientlybackground’ dissected, buttissue section before xMD. The general rule of thumb regardingcounte IHC 3,3 mogen slide charged chro a withstained IHC anddetails) morePROCEDURE on for (see mounted micro be should The cryosections ethanol. or or tome- paraffin- formalin from with or fixed tissue tissue cells frozenembedded recover to of used sections histological be from can xMD dissection, laser immunohistochemistry. standard and preparation Slide whole slides. of facilitate the rapid irradiation their software ferred motorized stages and because auto-selection we instruments tested, the Arcturus dissection commercial the Among modifications. minimal with handheld laser device. The specific goal was to adapt xMD to them three commercially available laser dissection instruments and on a In this paper, we protocolsdescribe for using xMD on technology design Experimental assess each and variable the select proper strategy.dissection pilot of tests on their samples before commencing a large study to NIH, set at a investigators recommend weroutinely that perform specimens related to tissue processing. In the LCM Core Laboratoryof quality the and dissection for available tissue the in cells target interest, of number of the molecule(s) the of level abundance the analysis, the of goals the of basis the on study particular their for the upstream tissue processing steps. during occurs that acids nucleic of fragmentation the of because sections) frozen for used that (beyond required often more,is or magnitude of order an of material, starting in increase stantive mRNA measurements from archival FFPE and samples microRNA in which be a to sub likely is xMD for niche unique The study. dissection laser standard a in procured cells of number small the Box 2 tory results. Ingeneral, the following features are optimal for xMD: Depending onthe tissuetype, target celland specifics of the dissection process, different EVA films may be required toobtainsatisfac is a translucent EVA membrane containing 19% vinyl acetate; it has a thickness of 76.2 this source has been discontinued and therefore we are currently using a CoTran membrane (3M, cat. no. 3M CoTran 9715). CoTran 9715extruded onto a paper-release liner as a thermoplastic seam tape (Electroseal) with an optimal heating temperature of 65 °C. However, ties of EVA film make it suitable for laser microdissection EVA is a copolymer of ethylene and vinyl acetate that has several medical and pharmaceutical applications. The thermoplastic proper Ultimately, investigators need to select the proper dissection tool

• • •

with higher melting temperatures generally increase the specificity of microdissection. A concentration of vinyl acetate~20%.The concentration of vinyl acetate correlates withmelting temperature. Inourtests, films Optical translucency. Film thickness 50–100 r stained afterward and no cover slip should be placed on the ′ daioezdn (DAB)-diaminobenzidine | EVA FILMF µ m. Note thatdissection of small targets may befacilitated with thinner films. O To effi improve dissection cell - XT R bearing jet near the laser diode and Veritas systems are pre 1 1 x Te ld sol nt be not should slide The . MD

instrument at NIH, iia to Similar 1 4 . For xMD, we initially used a 100------

tested, including power levels and duration of the laser pulse. pulse. laser the Overall, xMD of can be performed with settings available duration in all tested and levels power including tested, parameters. Laser to the film, resulting in a dissection. high-resolution With ( a relatively AE1/AE3 low laser power, cytokeratin target cells of were precisely targeting bonded using epithelium ( receptors estrogen against tive capture breast of cancer cells antibodies with immunolabeled and vary time depending on and the composition of the temperature EVA optimal film (see PROCEDURE the although min, 2–5 for the EVA polymer include a temperature ranging from 60 °C toadditional 80details). °C The best heating conditions for pretreatment of means to ensure close contact with the tissue (see PROCEDURE for roller, a using pressure gentle applied of proved optimal the to be ity of EVA from heating the polymer films, combined with the use pressure, and second, application of heat. The increased malleabil of EVA/tissueapplication first, the improve contact: to strategies devices without making substantive changes in them, we tested two wanted to adapt xMD technology to standard laser instruments and overlaying the the tissue with cells to be dissected. However, film as we the of contact enhance mechanically to served that output positive epithelium from EFPE prostate tissue ( tissue prostate EFPE from epithelium positive ( tissue brain rat frozen from nuclei individual we recovered NeuN-stained tissue, above step described to enhance the contact the between film and an is the ( tissue cells within stained This darkly heat to Avance. xMD for ‘gun’adapted laser Biotechnique wavelength nm 808 from system SDL-15 able plastic cap for is still necessary successful dissection. however,the laser; IR the of adjustment focus manual have not and placed on the slide for xMD. The Arcturus laser, in conjunction with a Macro Cap stripped of its original film IR the of adjustment and Veritasfocus II manual require PixCell parameters for xMD using LCM systems range from 30 to 75 mW. laser optimal general, In PROCEDURE). (see cells target of type the on dependent settings power with dissection, laser ventional 15 and 50 ms) and a higher overlap (50%) of the pulses than in con instruments, usually requiring longer pulse duration times (between to be performed for the first time in an instrument-free, ‘kit-like’ instrument-free, an in time first the for to performed be dissections allows system laser inexpensive relatively a of use the be completed (10–15 min), as well as precision (nuclei). Moreover, experiments demonstrate the speed at which xMD dissections can For the handheld laser device, we used a commercially avail commercially a used we device, laser handheld the For Box 2 Box ). As shown in in shown As ). µ Fig. 3c Fig. m and an optimal heating temperature of 75 °C. For each laser source, were parameters various µ m-thick DuPont ELVAX 410 EVA polymer , natureprotocols d ) and procured cytokeratin AE1/AE3- cytokeratin procured and ) Figure Figure Fig. 2a Fig. Fig. 3a 2 – , this approach allowed selec allowed approach this , e ) and dissection of prostate prostate of dissection and ) , b ). By the using premelting

| VOL.6 NO.4VOL.6 XT instrument does instrument protocol Fig. 3e Fig. | 2011 Fig. 2f Fig. , f ). These These ). - |

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© 2011 Nature America, Inc. All rights reserved. REAGENTS M often ones (smaller structures or cells target of size the tissue, of determined, as optimal laser parameters are dependent on the type be to need xMD for conditions specific Next,the samples. all on involving multiple institutions, testa scrape should be performed ences in among quality biomolecule them, such as a clinical study differ be could there that believe to reasons and specimens ple multi are there When needed. cells of number approximate the sue is compatible with the analysis method and further determining assay(s) to be used in the study, evaluating whether the stained tis the to according content the analyzes and buffer extraction into fromand slide controlfrom a nonstained section an IHC-stained procedure. The investigator simply scrapes a small amount of tissue immunostaining the and embedding) and procurement,fixation evaluating the effects of both upstream tissue processing (specimen step, first a as after IHC section tissue thereby the in quality ecule biomol assessing recommend Core,LCMwe NIH ments.the At the same way as investigators do when using standard laser instru samples and to determine optimal conditions,dissection in much tant to conduct pilot tests to evaluate the status of biomolecules in Pilot tests and controls. needs. specific their to meet eters) the protocol (laser pulse duration and power; film contact par of to adapt amount may need investigators and version this least with experience the have we thus, technology; the of iteration However, it should be noted that handheld xMD is the most recent bench by using xMD laboratory films and a low-cost source.light any on performed be can protocol the words, other In manner. film using the Veritas instrument. Magnification, ×100; scale bar,AE1/AE3-stained 100 EFPE prostate epithelial tissue that was transferred ontoinstrument. EVA Magnification, ×100; scale bar, 100 epithelial tissue that was transferred onto EVA film using the×100; PixCell scale II bar, 100 transferred. No stromal cells or unselected areas were captured.polymer Magnification, from the same area shown in arrows) remain on the slide. Magnification, ×100; scale bar,negative 100 stroma (black arrows) and cancer cells that were not irradiatedpositive for(white estrogen receptor that were captured using xMD. Note 500that tumor cells within areas that were not irradiated. Magnification, EVA×20; scalefilm. bar, However, the film did not capture the non-stainedThe tumor cellscells, positive stroma for estrogenor the receptor are selectively captured onto( the xMD (white arrows) remains intact. Magnification, ×20; scale bar, 500 and remain on the slide. Further, the tumor area that was notsuch irradiated as stroma for and fibroblasts (demarcated by black arrows) were not capturedonly estrogen-positive cells were microdissected, whereas negative regions after xMD and removal of the EVA film. Note that, within the irradiatedMagnification, area, ×20; scale bar, 500 Arrows indicate a tumor area that was intentionally not irradiated by linesthe waslaser. irradiated using the xMD protocol with an Arcturus cover slip and the tissue is covered by EVA film. The area demarked(brown by bluenuclear staining) without counterstaining. The slide does not havebreast a carcinoma. The tumor cells are immunolabeled for estrogen receptors( Figure 2 460 • c a protocol specimens sectioned at a thickness of 4–12 at athickness of sectioned specimens Frozen, ethanol-fixed or formalin-fixed, (FFPE) tissue paraffin-embedded ) Low-magnification view of the same EVA film in panels ) Low magnification view of a FFPE tissue section showing a high-grade ATER

µ | m. ( VOL.6 NO.4VOL.6

I | d ALS Examples of xMD using commercial microdissection instruments. ) Higher magnification view of the area of cancer cells (from panel | 2011 µ m. ( f | ) Anti-cytokeratin AE1/AE3-stained EFPE prostate

natureprotocols At the outset of an xMD study, it is impor µ m. ( Figure 2 b ) The same breast carcinoma section µ d m and mounted on glass slides m andmounted on glass . Only IHC-positive cells were µ m. ( g ) Anti-cytokeratin a and XT instrument. µ b m. ( after xMD. e µ µ ) EVA m. m.

m b ) ------

new lines of investigation of cells in histological sections. histological in cells of investigation of lines new many up opening advancement, exciting an be would resolution ments. Certainly, the evolution of xMD to technology this level of ele cellular minute procure to needed force the altering by goals to these achieve may steps be necessary pretreatment histological or even discrete molecular complexes, the although use parallel of organelles cellular small of level the to perhaps possible, be may overlying EVA suggests that improvements in precision dissection (see properties different with or sources alternative from polymers) other EVA (or using by them on improve to able be fact in may investigators should not feel constrained by these parameters, and used the specific types and sources of EVA film described; however, with especially respect to precision.dissection In this protocol, we development, further for area exciting an is films EVApolymer new of exploration the example, second a As protocol. the from deposition stain and radicals oxygen free by removing procedure deleterious effects the of immunolabeling, reduce as to this will simplifyapproach the IHC promising a is nanoparticles absorbing heat- with dyes colorimetric of replacement future example, one As technique. the to access gain community research the of bers are that to likely be improved on xMD technology as more mem directions. Future Protocols use established controls, e.g., those described previously in Finally, downstream molecular analysis of the dissected cells shouldpattern. staining IHC specific the and power) laser lower require tissue should be mountedtissue shouldbe on charged slides to prevent detachment during charged slides andstored at  f d a

CR Box 2 I T I CAL by Erickson ). The extreme by fidelity dyes which irradiated activate Frozen onto sectioned shouldbe positively tissuesections Looking forward, there are several aspects of of aspects several are there forward, Looking et et al. b

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CR I T I CAL Sections from Sections FFPE c Nature - - © 2011 Nature America, Inc. All rights reserved. • • • • Magnification, ×40; scale bar, 250 than 10 min using the handheld laser. of epithelial cells was microdissected in less AE3 targeting. An entire histological field cells procured by xMD using anti-cytokeratin AE1/ 250 tissue section. Magnification, ×40; scale bar, cytokeratin AE1/AE3-stained human prostate ×100; scale bar, 100 of xMD for certain applications. Magnification, the high-resolution microdissection capability were transferred to the EVA film demonstrating SDL-15 laser. IHC-positive individual nuclei entire tissue section was irradiated using the were targeted using NeuN antibody and the The nuclei of neurons from the granular layer NeuN-positive nuclei from frozen rat cerebellum. 250 cerebellum section. Magnification, ×40; scale bar, ( at a distance of 2–3 cm from the slide surface. The laser gun is pointed at an angle of 45° (CoTran 9715 EVA) and placed on a support. diode for xMD. The slide is covered by EVA film showing the positioning of the SDL-15 laser must be used (see However, appropriate laser safety precautions IHC-labeled cells in a relatively low-cost format. The instrument allows for rapid microdissection of laser diode device used for handheld xMD. Figure 3 • c DAB enhancer (Dako, cat. no. S1961) use. at 4°Cuntil Dako, cat. no. K4007). andDAB antibody Secondary reagents labeling (Dako EnVision Plus kit, backgroundAntibody reducer diluents with (Dako, cat. no. S3022) interest. dilutionantibody for each of target individual ing; therefore, determine the optimalprimary theuser shouldempirically Millipore, cat. no. MAB377). NeuN for frozen tissue(mousemonoclonal, brain rat dilution 1:1,000; (mouse monoclonal,specimens dilution 1:50; Dako, cat. no. M7047)and 1:50; Dako, cat. no. M3515), estrogen receptor for human breast cancer AE1/AE3 for human cells epithelial (AE1/AE3, mousemonoclonal, dilution to develop the current protocol cytokeratin included against antibodies for immunolabelingcells target antibodies for xMD.Primary Reagents used used. be to body anti particular the of sheet data the with conditions optimal the Check 9). or 6 (pH buffer citrate of use the as such conditions, retrieval antigen antibodies. cat. primary no. of themajority results S1699)provides with satisfactory binding. solution buffer (Dako, HIERusingcitrate or retrieval target antibody protein thetarget to expose theepitopenecessary for of primary reagents.Antigen retrieval For FFPE tissues, issometimes antigen retrieval takenbe unfixed with frozen samples. alab coat,including theuseof glovesglasses. care andsafety Extra should versal procedures, pathogen safety blood-borne tissue specimens, you shouldalways follow uni cut at8 typically suchorgans asbreast or prostate, are sections tively large. For from cell epithelial dissections ( sections small, whereas tissuemay brain require thick (4–5 thinner sections Forcell types. example, may lymphoma require depends on thetissueandtarget the section antibodies. procedure that isrequired for many primary the heat-induced (HIER) epitope retrieval ) NeuN immunostaining of a frozen rat µ µ m. ( m. (

| d f Handheld xMD. ( ≥ ) EVA film showing prostate epithelial ) An EVA film after xMD procurement of 12  

µ CR CR m), neurons because are rela I I µ Box 4 T T m. I I µ CAL CAL m. ( ! µ 

). ( m) as lymphocytes are m) aslymphocytes CAUT Proper thickness of Some primary antibodies may require special special require may antibodies primary Some

e CR a ) Image of an anti- b ) The SDL-15 ) Schematic diagram I I T  ON I CAL

CR When handling µ I m. Store your andDAB antibodies reagents T I CAL

xMDrequires strong IHCstain

- e c a

• • EQUIPMENT • • • • • • • • • • • • • Adhesive (Post-it pads Notes, 3M) HIER. Otheroptions include a pressure cooker or amicrowave. Black &Decker Handy Steamer (Black &Decker, cat. no. for HS1000) used (1.5-ml; tube Eppendorf) Eppendorf themomixerEppendorf (Eppendorf) (Electroseal) seamtape Thermoplastic DuPont ELVAX 410 EVA (DuPont) polymer Total protein isolation kit(ITSI Biosciences, cat. no. K-0011) cat. no. LC2676) Novex Tris-glycine SDSsample buffer (2×; Life Technologies, PicoPure kit(Life Technologies, cat. no. KIT0204) QIAamp DNA Micro kit(Qiagen, cat. no. 51304) formicrodissection used canbe xMD samples(see downstream analysis method. In general, thesamebuffers available for laser Molecular buffer. buffer extraction depends on extraction the of type The extraction. andtoxic.harmful Always proper with inside usexylenes air afumehood Xylenes (Sigma-Aldrich, cat. no. 247642) proper with inside airextraction. afumehood cat. no. E7023) Ethanol (95and100%(vol/vol), Sigma-Aldrich, grade; molecular Deionized (DI)water PBS (1×) !

CAUT I ON d b f Ethanol istoxic. Always solution useethanol

natureprotocols SDL-15 Glass slide !

CAUT Support I

| ON VOL.6 NO.4VOL.6 CoTran 9715EVA Box 1 Xylene is vapor protocol ).

IHC tissue | 2011

461 © 2011 Nature America, Inc. All rights reserved. 3| instruments. than the width of the slide. This is important to ensure that EVA on the slide fits in the slide holder of the automated 2| ? iccant, avoiding direct light exposure. The slides can be used safely within 1 week.   the stained tissue section.  must be obtained according to institutional review board protocols, including informed consent from subjects. ! 1| S PROCE • • • • • • 462 dyed filmthat be must removedwith forceps before xMD(see cat. no. LCM0212) Laser capture microdissection (LCM) Macro caps(Life Technologies, similar) Microscope-mounted Q-color3 or (Olympus (Olympus) camera digital evaluate EVA after polymer xMD. ×4, ×10and×20objectives and to used to verify IHCstainingspecificity A standard opticalmicroscope, BX41(Olympus), such astheOlympus with membrane, see 3Mor similar; Transparent ethylene vinyl acetate (EVA) film(3M CoTran 9715 EVA Scissors, forceps andfresh razor blades. rollerSealing (Bio-Rad, cat. no. MSR-0001) protocol

lide preparation for xMD

Box 4 Box 3 xMD device isan‘open’lasersource and thus requires the following safetymeasures: therefore, no additional safetyprecautions are necessary for the expression microdissection (xMD)protocol. However, the handheld All the commercially availablelaserdissection instruments described here (Arcturus caps contain adyed filmthat must be removed before xMD.The following simpleprocedure isused: the tissue. Thisisachieved withoptical plastic backing of aMacro LCMcap(LifeTechnologies). However, commercially availableLCM When performing xMDusing the Arcturus CAUT TROU

PAUSE CR CR

• • • • • • • • • • • • | VOL.6 NO.4VOL.6 With forceps, youshould carefully placethe trimmed EVA polymer onthe tissuesection. With a razor blade, cut a section of EVA film that is sufficient to cover the tissue section, but that is slightly narrower Stain the slide byIHCusing aprimary antibody of choice. Anexample IHCprotocol isprovided in

I I T T D I Before you usethe handheld laserdevice, a lasersafetyrepresentative should evaluate the workenvironment. The workarea should befree of reflective surfaces thatcouldcauseaccidental exposure. safety dome byLaservision (cat.no. 1307,dome filterpink(OD An acrylic box structure should beusedto house the laserand the slide toprevent accidental exposure. One example isalaser All windows and other openings of the room must becovered toprevent accidental exposure toindividuals. The lasershould behoused inalockable room withappropriate ‘warning’ signage. All usersshould alsowearlabcoatsand latex gloves. 808-nm range. All usersmust wearappropriate-wavelength laser safetygoggles withanoptical density (OD)valuehigher than7inthe Note: the prepared capcanbereused during the xMDprocedure. Carefully replace the capinthe original plastic tray. Clean the plastic surface of the capwithlens papersoaked in70%(vol/vol)ethanol. With cleaned forceps, pullthe plastic film from the cap. While youare wearing gloves, carefully remove acapfrom itsplastic tray. I I B ON URE CAL CAL LES

PO All animal tissues must be obtained according to relevant guidelines and regulations. All human tissue samples | |

H I STEP STEP LASERSAFETYPRECAUTI PREPARATI NT OOT | 2011  If DNA extraction is to be performed, the stained slides can be stored in a tightly closed jar containing des If RNA or proteins are to be extracted, we recommend performing xMD immediately after IHC staining. Do not counterstain the tissue with hematoxylin or other stains following IHC. Do not place a cover slip on

I CR N I G | T natureprotocols I CAL Box 2 The commercially The available capscontain a

● )

T O IMI N N O G 2h20minto1d(dependingonincubationtime) XT F AMACR , Veritas orPixCellIIsystems, the lasermust reach the proper focal plane atthe surface of Box 3 O O NS ). LCM CAPF

• • • • • • • •

> Lens paper PAP pen procedures to protect theoperator andlabpersonnel (see SDL-15The laser system andrequires harmful canbe safety additional Handheld, laser diodesystem (SDL-15, Biotechnique Avance) IIlaserPixCell capture (Life Technologies) microdissection instrument Veritas (Life laserTechnologies) microdissection instrument (Life Technologies) Arcturus Laser instrument. Four here options described are asfollows: (StatSpin, product number TS01; see Hot plate or hybridization chamber, such asStatSpinThermoBrite

7at755–830nm)). O XT R PERF laser microdissection instrument laser microdissection instrument XT , Veritas, PixCellII) haveenclosed lasers; O RMING Box 2 x ). MD

Box 5 . Box 4 !

CAUT ).

- © 2011 Nature America, Inc. All rights reserved. ? dissection instrument. Furthermore, the protocol canbeperformed using option D,ahandheld lasersystemthatdoes not require alasermicro 8| xMD process ? to xMD.  7| ?  6| ? film used (see  5|  4|

Box 5 21. 20. 19. 18. 17. 16. 15. 14. achieved before 5min. 13. 12. 11. may bemonoclonal (mouse) orpolyclonal (rabbit); hence, verifythatthe appropriate secondary antibody isused. 10. 9. specific antibody and antigen abundance. Refertothe data sheet provided bythe manufacturer asastarting point. 8. 7. 6. activity. with solution. The incubation time may need tobeadjusted, aseachtissuetypecontains different levelsof endogenous peroxidase 5. Do not useanexcess of PAP pen,asitmay spread onto the tissueand compromise IHCstaining. 4. Transfer the slide toDIwater. 3. the slides cooldown toroom temperature (RT)and transfer them toDIwater. preheated for 20mininaBlack&Decker steamer. Carefully placethe slides inside the heated target retrieval solution for 30min.Let 2. grades of ethanol (100%,95%and 70%(vol/vol))and then withDIwater. 1. EVA filmadjacent tothe target cells. Atypical IHCstaining protocol follows these general steps: Performing xMDrequires the useof achromogen such as3,3 TROU TROU TROU TROU

xMDcanbeperformed using option A(Arcturus CR CR CR CR

Wash the slide in1×PBS(30s, three times). Apply primary antibody overthe tissuesection. The antibody titer, incubation time and incubation temperature depend onthe Transfer the slide to1×PBS. Wash the slide inaDIwaterbath. Using the Dako Envision Pluskit,applyperoxidase blocking solution (bottle1inthe kit)for 10min.Cover the tissuegenerously Use aPAP pentodemark athinhydrophobic barrier around the tissuetoconcentrate the incubation solution overthe tissuearea. If frozen sections are used, thawthe slide immediately before staining and placethe section in70%(vol/vol)ethanol for 1min. Once the tissueishydrated, proceed withanantigen retrieval stepifnecessary. ForHIER,use1×Dako target retrieval solution For FFPEtissue, remove the paraffin wax from the histological section withaxylene bathand hydrate the tissuewith decreasing Allow the slide withthe EVA filmtocoolat room temperature (20–25°C) for 5min.The slide is now ready for xMD. Apply the roller tothe EVA polymer surface using gentle and uniform pressure. Place the slide withthe EVA filmona hot plateorinto a hybridization chambersetat60–80°C for 2–5min. Apply the sealing roller withuniform pressure across the EVA film onthe slide. I I I I Let the slide airdry inafume hood. Donot apply counterstaining ora coverslip.The slide isnow ready for xMD. Place the slide inxylene for 3min. Place the slide in100%ethanol bathfor 2 min (twotimes). Place the slide in95%(vol/vol)ethanol bathfor 2min(twotimes). Place the slide in70%(vol/vol)ethanol bath. Wash inDIwaterbath. Apply Dako DAB enhancer onthe slide for 3minatRT. Wash the tissueinDI waterbath(30s, twotimes). Apply the DAB solution onthe slide for 5minatRT. The usershould monitor thisstepcarefully, assufficient staining may be Prepare the DAB solution (one drop of DAB in1mlof DAB substrate solution; bottle3). Wash the slide in1×PBS(30s, three times). Using the Dako EnVision Pluskit,applythe secondary antibody (bottle2)for 30minatRT. Beaware thatthe primary antibody T T T T I I I I B B B B CAL CAL CAL CAL LES LES LES LES |

H H H H STEP STEP STEP STEP IMMUN ● OOT OOT OOT OOT Box 2

T IMI Because the EVA film is warm and malleable, contact between the film and the tissue is further improved. The optimal heating temperature needs to be predetermined by the user and depends on the type of EVA Once the EVA filmhasadhered tothe slide, itisour experience thatitisbesttoproceed immediately Thisisnecessary toensure good contact betweenthe EVA filmand the tissue. I I I I N N N N G G G G ). N G 10–60 min per slide, depending on the laser system used O HIST O CHEMISTRY F XT ), option B(Veritas) oroption C(Pixcell II)asdescribed below. ′ -diaminobenzidine (DAB) thatabsorbsthe laserenergy and activatesthe O R x MD

natureprotocols

| VOL.6 NO.4VOL.6 protocol | 2011

463 © 2011 Nature America, Inc. All rights reserved. ( (B) xMD with the Veritas microdissection system ( 464 (viii) (viii) protocol C A (vii) (vii) (vii) (iii) (iii) (iii) (vi) (iv) (xi) (ix) (vi) (iv) (vi) (iv) ) ) xMD with the (ii) (ii) (ii) ) ) xMD with the (v) (x) (v) (v) (i) (i) (i)

| VOL.6 NO.4VOL.6 control panel, placeacaponanarea of the slide containing IHC-positivecells. however, preliminary testsare needed tofind the optimal laserparameters for eachparticular application. targets require smaller spot size, lowerlaserpowerand lessduration thanlarger targets such as large epithelial cells;  duration between3and 10ms, laserpowerbetween 45and 75mW, and 50%spotoverlap. slide withthe filmbyclicking onthe slide holder.    control panel, openthe Veritas door and remove the slide withthe EVA filmfrom the Veritas. larger thanthe ×10field of view onthe Veritas. Multiple ×10fields are acceptable. selected. xMDwillcapture onlyIHC-positivecells. To avoid errors inthe Veritas software, do not selectasingle area  capture. down the light intensity. adjustment. area under the microscope and placethe same capunder the new region center. RepeatStep8A(v). cells; however, preliminary testsare needed tofind the optimal laserparameters for eachparticular application. finer targets require smaller spotsizes, lowerlaserpowerand less duration thanlarger targets such aslarge epithelial  size 50 Using the capture lasercontrol panel, youshould locateand focus the capture laser. Check thatthe fieldisstillonthe area withaclearspaceunder the cap.Then, withthe Veritas control panel, turn With the microscope control panel, selectthe ×10objective. Using the Veritas microscope control panel, youshould selectanarea under the capwithaclearspacefor laserfocus The Veritas instrument willautomatically take a‘road map’ image of the loaded slide. Thereafter, with the Veritas Load the modified xMDcaps(see Using the Veritas control panel, youshould openthe instrument and loadthe prepared slide withthe EVA filmonto the Once finished, remove the capfrom the slide by moving the captothe quality control position. Thereafter, remove the Repeat the process until allareas of IHC-positivecellsare dissected. If another area of the stained tissueistobemicrodissected, using the instrument controls, youmust selectthe new Using the Arcturus Using the Arcturus With the instrument controls, placethe caponanarea of the slide containing IHC-positivecells. Load the modified xMDcaps, which lackthe original dyed film(see Place the prepared slide withEVA filmonthe slide holder of the instrument. Activate the lasercontrol inthe PixCell IIcontrol box. Gently press the metallic capholder against the slide manually. Using the metallic armwiththe capholder, youshould remove one capand placeitonthe selectedarea of the slide. Activate the vacuumcontrol inthe PixCellIIcontrol box. While looking atthe screen orthrough the microscope eyepieces, youshould selectanarea withIHC-positive cells. Place the modified xMDcaps (see Place the prepared slide withEVA film on the microscope platform of PixCellII. Once youare finished, remove the capfrom the slide by moving the captoquality control position. Then, withthe If there isanother area of the tissuewithIHC-positivecellstobemicrodissected, withthe instrument controls, select In the Veritas control software, selectthe region tobeirradiated bycapture laserand then applythe command for With the capture lasercontrols, setupthe IRlaser according tothe following parameters: spotsize50 the new area under the microscope and placethe same capunder the new region. RepeatStep8B(ix).  can beselected. xMDwillonly capture IHC-positivecells.  IHC-positive cells, and then applythe command for capture.

CR CR CR CR CR CR CR CR I I I I I I I I T T T T T T T T I I I I I I I I µ

CAL CAL CAL CAL CAL CAL CAL CAL | 2011 m, laserpulseduration between30and 50ms, laserpowerbetween30and 75mW, and 50%spotoverlap.

P A STEP STEP STEP STEP STEP STEP STEP STEP ix rcturus |

C natureprotocols ell ell II laser capture microdissection instrument Optimal laserparameters depend onthe typeof tissue, target celland IHCmarker. Ingeneral, finer Donot usethe cutting laseratany point inthe xMD procedure. The Veritas instrument requires user-dependent focusing of the capture laserbefore xMD. The optimal laserparameters depend onthe typeof tissue, target celland IHCmarker. Ingeneral, Thisstepimproves the contact of the filmwiththe tissuesection. The whole fieldorthe area under the cap,including bothpositiveand negative cells, canbe Donot usethe cutting laseratany point inthe xMDprocedure. The whole fieldorthe whole area under the cap,including bothpositiveand negative cells, XT XT control software, youshould selectthe region to beirradiated bythe capture laser, including capture lasercontrol, youshould setupthe systemaccording tothe following parameters: spot X T microdissection system Box 3 Box 3

present stage ) into the capholder. ) onthe microscope captray.

buttoninthe Arcturus

Box 3 ). XT control panel.

µ m, laserpulse

© 2011 Nature America, Inc. All rights reserved. Troubleshooting advice canbefound in ? ? stored. For example, the PicoPure™ RNA kit (Life Technologies) allows the extracted sample to be stored at proteomics downstream analysis methods can be employed with the extracted biomolecules, including DNA methylation analysis  pieces of film. Check the manufacturer’s instructions regarding the proper incubation time (see and place the pieces inside a 1.5-ml Eppendorf tube containing the proper extraction buffer in a sufficient amount to cover the 13| ? application of the adhesive side of a sticky note pad (e.g., Post-it note) section or in the presence of cellular necrosis. Similar to standard laser dissection, off-target debris can be removed with the off-target debris, particularly when the tissue is excessively dried after staining, when folds are present in the tissue  12| standard microscope. 11| 10| 9| R (D) xMD procedure with a handheld laser (viii) (xii)

emoval and incubation of (iii) (iv) (xi) (ix) TROU TROU TROU

(ii) (x) PAUSE CR

(i) Once the presence of IHC-positive cells transferred onto the EVA film has been confirmed, cut the film with a razor blade Iftissuedebris isdetected onthe EVA polymer, applyanadhesive padtothe EVA toremove any unwanted material. Placethe EVA filmona new glassslide (without any tissue)toverifythe presence of procured IHC-positivecellsusing a Remove the EVA withforceps from the histological slide inone rapid motion. After completing xMDfollowing any of the previous options, youshould placethe slide withEVA filmonthe bench. I system and compromise dissection.  of the EVA film. two (‘2’)setting for laserintensity (5.7Jcm  amount of light tofocus the laser. ! SDL-15 diode isusedasalasersource. Placethe prepared slide withthe EVA inside asafetybox (see T from the microscope platform.   previous steps. cells; however, preliminary testsare needed tofind the optimal laserparameters for eachparticular application. fine targets require smaller spotsize, lowerlaserpowerand less duration than do larger targets such aslarge epithelial  100 mW;pulseduration, 15–50ms; repeat Irradiate the tissuecovered byEVA filmwith~1,200laserpulses. Hold the SDL-15lasergunata45°angle tothe slide surface and keep itbetween2and 3cmabovethe surface Set the SDL-15instrument tothe number three (‘3’)position for pulsefrequency (7pulsespers)and tothe number This option does not require the useof alasermicrodissection instrument. Instead, ahandheld lasersuch asthe Once youare finished, remove the capfrom the slide using the metallic arm.Then, remove the slide withthe EVA film If another area of the tissuealsocontains IHC-positivecells, move the captothe new area and repeat the Irradiate the entire area under the cap,including IHC-stained and unstained cells. With the PixCellIIcontrol box, adjustthe laseraccording tothe following parameters: laserpower, between30and Using the screen, youshould find acleararea under the cap. Move the microscope tothe ×10objectiveand reduce the 

I B B B CAUT

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LES LES LES CR CR CR CR CR CR

PO 2 I 6 I I I I I

and RT-PCR T H H H T I T T T T STEP I NT I I I I I I OOT OOT OOT ON CAL CAL CAL CAL CAL CAL The molecular extraction procedure is performed using standard protocols for LCM samples. A number of Wear alabcoat,glovesand lasersafetyglasses(see In general, xMD is very specific for target cells; however, occasionally, you will observe the presence of I I I

N N N STEP STEP STEP STEP STEP STEP G G G

Donot allowthe tipof the laserguntocontact the surface of the film,asthis may damage the The xMDprocedure onthe PixCellIIrequires fine laser focus. When irradiating the EVA, awetting appearance isobservedwhen the filmissuccessfully activated. The number of laserpulsesdepends onthe sizeof the tissuetobeirradiated. The optimal laserparameters depend onthe typeof tissue, target celland IHCmarker. Ingeneral, 2 OnlyIHC-positivecellsare captured. 7 . Depending on the molecule and the kit or protocol employed, the cell extract can be temporarily E V A film after xMD T

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= ). T

0.2s;target IMI N G 10 min or until molecular extraction process is complete

0.300V;spotsize Box 4

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| VOL.6 NO.4VOL.6

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80° C until use. protocol ).

12,13,22 2011

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465

© 2011 Nature America, Inc. All rights reserved. and M.R.E.-B. wrote the manuscript. J.C.H. and M.A.T. contributed equally J.R.-C., M.A.T. and M.R.E.-B. analyzed the data. J.C.H., M.A.T., J.R.-C. experiments. J.C.H., S.K., M.D.A. and M.A.T. conducted the tests. J.C.H., expression microdissection (xMD). J.C.H., M.A.T. and M.R.E.-B. designed the AUT program of the NIH National Cancer Institute, Center for Cancer Research. A measurement (NanoDrop, Thermo Scientific) of the extracted biomolecules is recommended. tissue section atthe outsetof astudy toobtainanestimate of quality and quantity. Inaddition, spectrophotometer-based biomolecule recovery can benegatively affectedbythe IHCstaining process; thus, itisimportant toanalyze anIHC-stained or tissuedebris. The results of the dissection canbeevaluated using alight microscope. xMD filmshould contain the IHC-positivecellscaptured onthe filmwithout significant contamination byIHC-negative cells Typical xMDresults obtained witheachof the lasersystems are shown in ANT Step 13:variable, dependent onthe typeof extraction buffer(see Steps 9–12:10min Step 8D,handheld xMD:10–15min,depending onthe sizeof the tissuesection Step 8C,PixCellII:45–60min,depending onthe typesof tissueand target cell Step 8B,Veritas: 30–45min,depending onthe types of tissueand target cells Step 8A,Arcturus Steps 2–7: 15–20 min Step 1,Including ● T 466 to the work. a 13 12 8 (options B, C) 8 (options A–D) 5–7 1 S protocol cknowle

tep The yield of biomolecules depends onthe number of procured cellsand onthe method of tissuepreservation. However, b T H

le IMI | I OR VOL.6 NO.4VOL.6 C 1 I

CONTR PATE N | dgm G

Troubleshooting table. D IB ents UT RESULTS | 2011 I

This This research was supported, in part, by the intramural ONS XT Box 5 : 30–45min,depending onthe typeof tissueand target cells after after extraction No biomolecules detectable the film Too much tissue debris on Cells did not transfer to EVA Cells did not transfer to EVA Cells did not transfer to EVA Cells did not transfer to EVA P R.F.B., T.J.P., M.A.T. and M.R.E.-B. developed roblem | natureprotocols : 2htoovernight (depending onthe incubation time needed for the primary antibody). the film Buffer did not cover Slide too dry focused Laser was not properly Laser setup not adequate prepared EVA was not properly dehydrated The slide was not properly P ossible reason

1. com/reprintsandpermissions/. http://npg.nature. at online available is information permissions and Reprints Published online at http://www.natureprotocols.com/. for details). (see HTML the version of interests article this CO

M Box 1 PET 998–1001 (1996). 998–1001 Emmert-Buck, M.R. Emmert-Buck, I N G G FI ) Figures 2 NANC the pieces of the film Increase the amount of extraction buffer to cover all pressure to remove the tissue debris Apply an adhesive pad on the EVA with gentle ethanol and xylene baths ( Decrease the length of time in the final 100% Focus the capture laser before xMD duration Test a different laser setup: increase power and/or Apply the roller on the EVA with more pressure Increase the time of heat incubation of the EVA film Use fresh ethanol solutions ethanol and xylene baths ( Increase the length of time in the final 100% S Test a different extraction buffer Use a shaker or themomixer for incubation olution I AL I et al. et NTERESTS and Laser capture microdissection. capture Laser 3 . Afterasuccessful dissection, the The authors declare competing financial declare financial competing The authors

Box 5 Box 5 ) )

Science

274

371–377 (2004). 371–377 specimens. tissue of profiling expressiongene for microdissection. capture (2006).13 RARbeta2promotermethylation humanprostate incancer. cells. stromal associated (2004). profiling. molecular for cells of retrieval 2010). October (11 doi:10.1001/archneurol.2010.261 tissue. nerve in type neuropathy (2009). spectrometry.mass biological and microdissection capture laser using proteomics tissue by basis samples. tissue microdissected Bioinformatics BMC models. tumor xenograft in expressiongene of analysis component Mitochondrion (m.5636T 2010). October (12 Res. Cancer Clin. cancer.breast in disease metastaticcorresponding and primary (2006). surgicalin pathology. tissue. Perlmutter,M.A. laser of modelingThermal Bonner,R.F. P.G.& McQueen, S.R., Goldstein, Grover,A.C. J.A. Hanson, Tangrea,M.A. C.J. Klein, D.J. Johann, H.S. Erickson, X. Yang, T.Pinós, J. Jensen, Dupont V.Espina, Hunt,Finkelstein,J.L.& S.D.Microdissection techniques formolecular testing Bonner,R.F. Science et al. et

et al. et > et al. et

C) in a patient with progressive external ophthalmoplegia.external progressive with patient a in C) et al. et et al. et et al. et et al. et

et al. et

Stromal microenvironment processes unveiled by biological by unveiled microenvironmentprocesses Stromal et al. et 11 et al. et A novel mutation in the mitochondrial tRNA(Ala) gene tRNA(Ala) mitochondrialthe in mutation novel A 278 Laser-capture microdissection. Laser-capture published online, doi:10.1158/1078-0432.CCR-10-1133 online, published Mass spectrometric-based proteomic analysis of amyloid of analysis proteomicspectrometric-based Mass et al. et , 228–233 (2011). 228–233 , Tumor-associated endothelial anddisplayGSTP1cells Laser capture microdissection: molecular analysis of analysis molecular microdissection: capture Laser

Approaching solid tumor heterogeneity on a cellular a on heterogeneitytumor solid Approaching Gene promoter methylation in prostate tumor- prostate in methylationpromoter Gene et al. et , 1481–1483 (1997). 1481–1483 , 11 Expression microdissection: operator-independentmicrodissection:Expression Quantitative RT-PCR gene expression analysis of laser of analysis expressiongene RT-PCR Quantitative Arch.Pathol. Lab.Med. Comparison of snap freezing versus ethanol fixation ethanol versus freezing snap of Comparison (Suppl 9), S11 (2010). S11 9), (Suppl PIK3CA mutations may be discordant between discordant be may mutations PIK3CA Appl. Opt. Appl. J. Natl. Cancer Inst. Cancer Natl. J. J. Proteome. Res. Proteome. J. Nat. Protoc. Nat. Arch. Neurol. Arch.

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6 ,

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45–51 (2010).45–51 transcriptase-polymerase chain reaction.transcriptase-polymerase chain reverse real-time andmicrodissection laser in preservation mRNA high-quality for methodimmunolabelingultra-short novel a Validationof Ther. Biol. Cancer microenvironment. tumor the of constituents cellular of profile expression 394–401(2010). methylationDNAfor analysisarchivalof tissuespecimens. microdissection. capture immuno-laser on emphasis with applications and aspects methodicalmicrodissection: (2009). 187–193 samples. proteomic prepare to sections (2008). expressiongene barrier brain blood- probe to RT-PCR quantitative with coupled microdissection capture cancer.prostate microenvironmentin (1999). 61–66 analysis. mRNA for sections frozen immunostained (2003). studies.profiling molecular for specimens valuable tool for risk assessmentriskvaluableforprevention andtool trials. expressionlaser-captureanalysisin microdissected prostatebiopsies: (2010). 2529–2544 ontology.gene and analysis enrichment set protein by annotationmicrodissection: capture laser using cells microdissection. laser and von Smolinski, D., Blessenohl, M., Neubauer, C., Kalies, K. & Gebert, A. Gebert, & K. Kalies,Neubauer, C., M., Blessenohl, D., Smolinski, von R.J. Buckanovich, Eberle,F.C. capture Laser L. Quintanilla-Martinez, & Kremer,F.,M. Fend, Nonn, L., Vaishnav,Nonn,L., P.H.Gallagher, micro-RNAGann,andA.,mRNA & L. S. Cha, immunolabeling for preservation RNA D.W.Improved Smith, & A.L. Brown, Liu, Y. Liu, immuno-laser Validationof Pachter,J.S. & N. Murugesan, J.A., Macdonald, J. Rodriguez-Canales, F.Fend, tissue clinical of J.W.HandlingGillespie, & M.R. Emmert-Buck, I.M., Leiva, et al. et et al. et et al. et et al. et Immuno-laser capture microdissection of frozen prolactioma frozen of microdissection capture Immuno-laser

Immuno-LCM: laser capture microdissection of microdissection capture laser Immuno-LCM: In situ In Immunoguided assistedlasermicrodissection techniques

5 et al. et , 635–642 (2006). 635–642 , proteomic analysis of human breast cancer epithelial cancer breast human of analysis proteomic et al. et Use of immuno-LCM to identify the identify to immuno-LCM of Use natureprotocols RNA Identification of a unique epigenetic sub- epigenetic unique a Identification of in situ in

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