Laser Capture Microdissection

Espina, Virginia; Milia, J; Wu, Glendon; Cowherd, Stacy; La, Liotta

doi:10.1007/978-1-59259-993-6_10

Cited by 76 publications

(76 citation statements)

References 19 publications

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“…The mounted tissue sections were left to dry overnight at room temperature (http://support.moleculardevices.com). To visualise the areas of interest, the tissue sections were stained for initial scrape tests with H&E, and for all other interventions with Cresyl Fast Violet according to [59] with slight modifications (Table 3). The slides were completely air dried before microdissection to allow for proper excision performance.…”

Section: Tissue Processing and Stainingmentioning

confidence: 99%

“…Extraction of mRNA was performed immediately after microdissection within three hours after staining, due to potential RNase contamination during storage [59]. A Recover All™ Total Nucleic Acid Isolation Kit for FFPE (Ambion™) was used to extract the mRNA according to the manufacturer's protocol with the following small adjustments.…”

Section: Isolation Of Mrna From Ffpe Tissue Sections Isolated By Lcmmentioning

confidence: 99%

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Analysis of Gene Expression Signatures in Cancer-Associated Stroma from Canine Mammary Tumours Reveals Molecular Homology to Human Breast Carcinomas

Ettlin

Clementi

Amini

et al. 2018

Journal of Comparative Pathology

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Cancer-associated stroma (CAS) plays a key role in cancer initiation and progression. Spontaneously occurring canine mammary carcinomas are viewed as excellent models of human breast carcinomas. Considering the importance of CAS for human cancer, it likely plays a central role in canine tumours as well. So far, however, canine CAS lacks characterisation, and it remains unclear whether the biology between CAS from canine and human tumours is comparable. In this proof-of-principle study, using laser-capture microdissection, we isolated CAS and normal stroma from 13 formalin-fixed paraffin embedded canine simple mammary carcinomas and analysed the expression of seven known human CAS markers by RT-qPCR (Reverse Transcription quantitative PCR) and validated some targets by immunohistochemistry. We found that Col1a1 (Collagen1α1), αSMA (alpha Smooth Muscle Actin), FAP (Fibroblast activation protein), PDGFRβ (Platelet-derived growth factor receptor beta), and Caveolin-1 were significantly upregulated in canine CAS, and the expression of CXCL12 (Stromal cell derived factor 1) significantly decreased, whereas MMP2 (Matrix Metalloproteinase 1) and IL6 (Interleukin 6) did not change. Our results suggest strong similarities in CAS biology in canine and human mammary carcinomas but also reveal some differences. To the best of our knowledge, this is the first report to provide a comprehensive expression analysis of the most important CAS markers in canine simple mammary carcinomas and further supports the validity of the dog as model for human cancer.

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Section: Tissue Processing and Stainingmentioning

confidence: 99%

Section: Isolation Of Mrna From Ffpe Tissue Sections Isolated By Lcmmentioning

confidence: 99%

Analysis of Gene Expression Signatures in Cancer-Associated Stroma from Canine Mammary Tumours Reveals Molecular Homology to Human Breast Carcinomas

Ettlin

Clementi

Amini

et al. 2018

Journal of Comparative Pathology

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“…More recently, combined with expressed fluorescent tags, LCM has been used to isolate small neurons from Drosophila brain sections (Iyer and Cox, 2010). Popular uses of LCM include DNA, RNA, and proteomics applications (Espina et al, 2006). Isolation of individual live cells from suspensions shows promise for applications of highthroughput single-cell and even organelle microanalysis.…”

Section: Sampling Strategies For Microanalysismentioning

confidence: 99%

Small-Volume Analysis of Cell–Cell Signaling Molecules in the Brain

Romanova

Aerts

Croushore

et al. 2013

Neuropsychopharmacol

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Modern science is characterized by integration and synergy between research fields. Accordingly, as technological advances allow new and more ambitious quests in scientific inquiry, numerous analytical and engineering techniques have become useful tools in biological research. The focus of this review is on cutting edge technologies that aid direct measurement of bioactive compounds in the nervous system to facilitate fundamental research, diagnostics, and drug discovery. We discuss challenges associated with measurement of cell-to-cell signaling molecules in the nervous system, and advocate for a decrease of sample volumes to the nanoliter volume regimen for improved analysis outcomes. We highlight effective approaches for the collection, separation, and detection of such small-volume samples, present strategies for targeted and discovery-oriented research, and describe the required technology advances that will empower future translational science.

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“…The LCM system is based on an inverted light microscope, which is fitted with a laser device for visualizing and procuring cells. The two main classes of LCM are infrared (IR) capture systems and ultraviolet (UV) cutting systems (Espina et al, 2006). LCM instruments (IR and IR/UV systems) are available in automated (robotic) and manual platforms.…”

Section: Basic Principles Of Lcmmentioning

confidence: 99%

“…The main limitation of LCM is the requirement for identifying cells of interest based on morphological characteristics (Espina et al, 2006). Cells are usually identified by a cytologist, pathologist, or technologist who is skillful in identifying cells.…”

Section: Disadvantages Of Lcmmentioning

confidence: 99%

Laser‐Capture Microdissection

Jensen¹

2013

The Anatomical Record

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BACKGROUNDLaser capture microdissection (LCM) is used to isolate specific cells from microscopic regions of tissue, cells, or organisms. LCM is also known as microdissection, laser-assisted microdissection, or laser microdissection. Various tissue microdissection techniques have been used to isolate pure cell populations. LCM was developed to address the problem of tissue heterogeneity, being a mixture of different cell types. Liotta and coworkers (1996) developed an LCM system to overcome the disadvantages of previous tissue microdissection techniques, such as manual microdissection and gross dissection of frozen tissue blocks to enrich for specific cell populations. They developed this system primarily for molecular analysis of solid tumors. The system was rapidly applied for commercial production by Arcturus Engineering (Mountain View, CA).Three different classes of biomolecules, DNA, RNA, and proteins, can be investigated using LCM specimens (Domazet et al., 2008). LCM technology can fulfill the needs of researchers for routinely performing tissue microdissection. This method has proven invaluable to molecular pathology research and is used in many laboratories worldwide. BASIC PRINCIPLES OF LCMThe basic principle of LCM is the capture of cells or an individual cell onto a thermoplastic membrane from sections of stained tissue (frozen or fixed and wax-embedded sections) or cytological preparations. Cellular components, including RNA, DNA, and protein, can be extracted using the appropriate methods and used for molecular analysis. The LCM system is based on an inverted light microscope, which is fitted with a laser device for visualizing and procuring cells. The two main classes of LCM are infrared (IR) capture systems and ultraviolet (UV) cutting systems (Espina et al., 2006). LCM instruments (IR and IR/UV systems) are available in automated (robotic) and manual platforms.For IR capture systems, a near-IR laser is attached to a microscope stage. This laser is applied for melting a thermolabile polymer film. A focused laser beam of varying diameter (7.5, 15, and 30 mm) is applied for localized melting of the film over the cells that have been selected. Laser impulses, which are usually 0.5-5 msec long, are able to be repeated, enabling rapid isolation of a large number of cells. The film is produced at the bottom of plastic cap of optical quality. This cap allows the laser to be focused in the same plane as the tissue section. The polymer film melts in the area where the laser impulse is present, resulting in a polymer-cell composite. Only those cells that are within the diameter of the melted polymer are targeted for microdissection with each laser pulse. When the polymer is removed from the surface of the tissue, the embedded targeted cells are sheared away from the tissue section. Cells are solubilized by adding extraction buffer, and the desired molecules are released. In LCM, there are physical forces involved, including an upward binding force between the tissue and substratum, lateral force between ce...

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Laser Capture Microdissection

Cited by 76 publications

References 19 publications

Analysis of Gene Expression Signatures in Cancer-Associated Stroma from Canine Mammary Tumours Reveals Molecular Homology to Human Breast Carcinomas

Analysis of Gene Expression Signatures in Cancer-Associated Stroma from Canine Mammary Tumours Reveals Molecular Homology to Human Breast Carcinomas

Small-Volume Analysis of Cell–Cell Signaling Molecules in the Brain

Laser‐Capture Microdissection

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