Abstract
Laser capture microdissection (LCM) is a technique that allows procurement of an enriched cell population from a heterogeneous tissue sample under direct microscopic visualization. Fundamentally, laser capture microdissection consists of three main steps: (1) visualizing the desired cell population by microscopy, (2) melting a thermolabile polymer onto the desired cell populations using infrared laser energy to form a polymer-cell composite (capture method) or photovolatizing a region of tissue using ultraviolet laser energy (cutting method), and (3) removing the desired cell population from the heterogeneous tissue. In this chapter, we discuss the infrared capture method only. LCM technology is compatible with a wide range of downstream applications such as mass spectrometry, DNA genotyping and RNA transcript profiling, cDNA library generation, proteomics discovery, and signal pathway mapping. This chapter profiles the ArcturusXT™ laser capture microdissection instrument, using isolation of specific cortical lamina from nonhuman primate brain regions, and sample preparation methods for downstream proteomic applications.
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References
Mueller C, deCarvalho AC, Mikkelsen T, Lehman NL, Calvert V, Espina V, Liotta LA, Petricoin EF 3rd (2014) Glioblastoma cell enrichment is critical for analysis of phosphorylated drug targets and proteomic-genomic correlations. Cancer Res 74(3):818–828. doi:10.1158/0008-5472.CAN-13-2172
Shipp S (2007) Structure and function of the cerebral cortex. Curr Biol 17(12):R443–R449. doi:10.1016/j.cub.2007.03.044
Gusev PA, Gubin AN (2010) Arc/Arg3.1 mRNA global expression patterns elicited by memory recall in cerebral cortex differ for remote versus recent spatial memories. Front Integr Neurosci 4:15. doi:10.3389/fnint.2010.00015
Kok P, Bains LJ, van Mourik T, Norris DG, de Lange FP (2016) Selective activation of the deep layers of the human primary visual cortex by top-down feedback. Curr Biol 26(3):371–376. doi:10.1016/j.cub.2015.12.038
Maass A, Schutze H, Speck O, Yonelinas A, Tempelmann C, Heinze HJ, Berron D, Cardenas-Blanco A, Brodersen KH, Stephan KE, Duzel E (2014) Laminar activity in the hippocampus and entorhinal cortex related to novelty and episodic encoding. Nat Commun 5:5547. doi:10.1038/ncomms6547
Maier A, Adams GK, Aura C, Leopold DA (2010) Distinct superficial and deep laminar domains of activity in the visual cortex during rest and stimulation. Front Syst Neurosci 4. doi:10.3389/fnsys.2010.00031
Schroeder CE, Foxe JJ (2002) The timing and laminar profile of converging inputs to multisensory areas of the macaque neocortex. Brain Res Cogn Brain Res 14(1):187–198
Takeda M, Koyano KW, Hirabayashi T, Adachi Y, Miyashita Y (2015) Top-down regulation of laminar circuit via inter-area signal for successful object memory recall in monkey temporal cortex. Neuron 86(3):840–852. doi:10.1016/j.neuron.2015.03.047
Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA (1996) Laser capture microdissection. Science 274(5289):998–1001
Simone NL, Remaley AT, Charboneau L, Petricoin EF 3rd, Glickman JW, Emmert-Buck MR, Fleisher TA, Liotta LA (2000) Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection. Am J Pathol 156(2):445–452. doi:10.1016/S0002-9440(10)64749-9
Boone DR, Sell SL, Hellmich HL (2013) Laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury. J Vis Exp 74. doi:10.3791/50308
Drummond ES, Nayak S, Ueberheide B, Wisniewski T (2015) Proteomic analysis of neurons microdissected from formalin-fixed, paraffin-embedded Alzheimer’s disease brain tissue. Sci Rep 5:15456. doi:10.1038/srep15456
Molina M, Steinbach S, Park YM, Yun SY, Di Lorenzo Alho AT, Heinsen H, Grinberg LT, Marcus K, Leite RE, May C (2015) Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis. J Neural Transm 122(7):993–1005. doi:10.1007/s00702-015-1414-4
Pietersen CY, Lim MP, Macey L, Woo TU, Sonntag KC (2011) Neuronal type-specific gene expression profiling and laser-capture microdissection. Methods Mol Biol 755:327–343. doi:10.1007/978-1-61779-163-5_28
Kolble K (2000) The LEICA microdissection system: design and applications. J Mol Med 78(7):B24–B25
Gallagher RI, Blakely SR, Liotta LA, Espina V (2012) Laser capture microdissection: Arcturus(XT) infrared capture and UV cutting methods. Methods Mol Biol 823:157–178. doi:10.1007/978-1-60327-216-2_11
Mueller C, Edmiston KH, Carpenter C, Gaffney E, Ryan C, Ward R, White S, Memeo L, Colarossi C, Petricoin EF 3rd, Liotta LA, Espina V (2011) One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens. PLoS One 6(8):e23780. doi:10.1371/journal.pone.0023780
Botling J, Edlund K, Segersten U, Tahmasebpoor S, Engstrom M, Sundstrom M, Malmstrom PU, Micke P (2009) Impact of thawing on RNA integrity and gene expression analysis in fresh frozen tissue. Diagn Mol Pathol 18(1):44–52. doi:10.1097/PDM.0b013e3181857e92
Espina V, Edmiston KH, Heiby M, Pierobon M, Sciro M, Merritt B, Banks S, Deng J, VanMeter AJ, Geho DH, Pastore L, Sennesh J, Petricoin EF 3rd, Liotta LA (2008) A portrait of tissue phosphoprotein stability in the clinical tissue procurement process. Mol Cell Proteomics 7(10):1998–2018. doi:10.1074/mcp.M700596-MCP200
Lim MD, Dickherber A, Compton CC (2011) Before you analyze a human specimen, think quality, variability, and bias. Anal Chem 83(1):8–13. doi:10.1021/ac1018974
Micke P, Ohshima M, Tahmasebpoor S, Ren ZP, Ostman A, Ponten F, Botling J (2006) Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens. Lab Invest 86(2):202–211. doi:10.1038/labinvest.3700372
Xiang CC, Mezey E, Chen M, Key S, Ma L, Brownstein MJ (2004) Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling. Nucleic Acids Res 32(22):e185. doi:10.1093/nar/gnh185
Wang H, Owens JD, Shih JH, Li MC, Bonner RF, Mushinski JF (2006) Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA. BMC Genomics 7:97. doi:10.1186/1471-2164-7-97
Mouledous L, Hunt S, Harcourt R, Harry J, Williams KL, Gutstein HB (2003) Navigated laser capture microdissection as an alternative to direct histological staining for proteomic analysis of brain samples. Proteomics 3(5):610–615. doi:10.1002/pmic.200300398
Wong MH, Saam JR, Stappenbeck TS, Rexer CH, Gordon JI (2000) Genetic mosaic analysis based on Cre recombinase and navigated laser capture microdissection. Proc Natl Acad Sci U S A 97(23):12601–12606. doi:10.1073/pnas.230237997
Buckanovich RJ, Sasaroli D, O’Brien-Jenkins A, Botbyl J, Conejo-Garcia JR, Benencia F, Liotta LA, Gimotty PA, Coukos G (2006) Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment. Cancer Biol Ther 5(6):635–642
Demarest TG, Murugesan N, Shrestha B, Pachter JS (2012) Rapid expression profiling of brain microvascular endothelial cells by immuno-laser capture microdissection coupled to TaqMan((R)) low density array. J Neurosci Methods 206(2):200–204. doi:10.1016/j.jneumeth.2012.02.023
Murakami H, Liotta L, Star RA (2000) IF-LCM: laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication. Kidney Int 58(3):1346–1353. doi:10.1046/j.1523-1755.2000.00295.x
Waller R, Woodroofe MN, Francese S, Heath PR, Wharton SB, Ince PG, Sharrack B, Simpson JE (2012) Isolation of enriched glial populations from post-mortem human CNS material by immuno-laser capture microdissection. J Neurosci Methods 208(2):108–113. doi:10.1016/j.jneumeth.2012.04.014
Espina V, Wulfkuhle JD, Calvert VS, VanMeter A, Zhou W, Coukos G, Geho DH, Petricoin EF 3rd, Liotta LA (2006) Laser-capture microdissection. Nat Protoc 1(2):586–603. doi:10.1038/nprot.2006.85
Suzuki WA, Amaral DG (1994) Perirhinal and parahippocampal cortices of the macaque monkey: cortical afferents. J Comp Neurol 350(4):497–533. doi:10.1002/cne.903500402
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Corgiat, B.A., Mueller, C. (2017). Using Laser Capture Microdissection to Isolate Cortical Laminae in Nonhuman Primate Brain. In: Espina, V. (eds) Molecular Profiling. Methods in Molecular Biology, vol 1606. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6990-6_8
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DOI: https://doi.org/10.1007/978-1-4939-6990-6_8
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