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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.

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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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