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Nat Protoc. Author manuscript; available in PMC Mar Published in final edited form as: Published online Mar Abstract Laser-based microdissection facilitates the isolation of specific cell populations from clinical or animal model tissue specimens for molecular analysis. Expression microdissection xMD is a second-generation technology that offers considerable advantages in dissection capabilities; however, until recently the method has not been accessible to investigators.
This protocol describes the adaptation of xMD to commonly used laser microdissection instruments and to a commercially available handheld laser device in order to make the technique widely available to the biomedical research community. The method improves dissection speed for many applications by using a targeting probe for cell procurement in place of an operator-based, cell-by-cell selection process.
Moreover, xMD can provide improved dissection precision because of the unique characteristics of film activation. The time to complete the protocol is highly dependent on the target cell population and the number of cells needed for subsequent molecular analysis.
INTRODUCTION Development of the protocol Since its original invention in the s, laser microdissection technology has evolved substantially, with the development of several commercially available dissection instruments and the incorporation of computerized platforms and image analysis software 1 — 4. In parallel, protocols for analyzing small amounts of DNA, RNA and protein have improved, facilitating quantitative and in-depth analysis of cellular genomes, transcriptomes and proteomes 5 — Current laser-based instruments have successfully advanced the molecular pathology field, and microdissection studies are now routinely used by a wide spectrum of laboratory- and clinic-based researchers.
However, this field has certain challenges, such as the need for improved precision and the requirement for relatively large amounts of biomolecules for some downstream analysis platforms. Expression microdissection xMD represents a conceptual and practical advance that offers substantive improvements for laser dissection based on operator-independent selection of cells using molecular targeting For xMD, a clear ethylene vinyl acetate EVA film is applied to a histological slide containing immunohistochemically IHC stained cells and exposed to a light source that irradiates the entire tissue section Fig.
The light energy is absorbed by the chromogen, causing focal heat activation of the dissection film and bonding it to target cells. Removal of the film from the histological section procures immunostained cells for subsequent molecular analysis.
Expression microdissection adapted to commercial laser dissection instruments
In this method, histological sections are first stained for cellular markers via either chemical or immuno-guided methods, placed in close contact with an ethylene vinyl acetate EVA film, and exposed to a light source. The focal, transient heating of the stained cells or subcellular structures melts the EVA film selectively to the targets for procurement. In this report, we introduce a custom-designed flashcube system that permits consistent and reproducible microdissection of nuclei across an FFPE rat brain tissue section in milliseconds. In addition, we present a method to efficiently recover and combine captured proteins from multiple xMD films. Both light and scanning electron microscopy demonstrated captured nuclear structures. Targeted mass spectrometry using Multiple Reaction Monitoring MRM showed more impressive data, with a 3-fold enrichment in histones, and a concurrent depletion of proteins localized to the cytoplasm, cytoskeleton, and mitochondria. These data demonstrate that the flashcube-xMD technology is applicable to the proteomic study of a broad range of targets in molecular pathology.
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