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Laser Capture Microdissection of Cells Labeled with Enhanced Green Fluorescent Protein

Vasker Bhattacherjee, Partha Mukhopadhyay, Saurabh Singh, Robert M. Greene and M. Michele Pisano, University of Louisville Birth Defects Center, Department of Molecular, Cellular and Craniofacial Biology, Louisville, KY 40202


Abstract
This application note describes a Laser Capture Microdissection (LCM) method for the isolation of cells expressing enhanced green fluorescent protein (EGFP) from frozen sections of embryonic tissue. The application of this methodology was used to facilitate retrieval of RNA from mouse embryonic neural crest cells that could not be distinguished by morphology, histological stains or labeling by fluorescent antibodies in a heterogeneous tissue. Specificity of the LCM of the neural crest cells was confirmed by quantitative real-time PCR of neural crest gene markers.


Introduction
The emergent technology of Laser Capture Microdissection (LCM; Arcturus; Emmert-Buck et al., 1996; Simone et al., 1998) is used to isolate specific cell types from tissue sections for downstream molecular analyses. In traditional applications, LCM is used to isolate cells that are distinguished by morphology, histological stains, or labeling by fluorescent antibodies from a heterogeneous population of cells in a tissue section. In this Application Note, an adaptation of this technique for the microdissection of cells genetically labeled with enhanced green fluorescent protein is described (EGFP; Bhattacherjee et al., 2004). EGFP is a recombinant protein originally derived from the jellyfish, Aequoria victoria, which has the advantage over other reporters of not requiring cell/tissue fixation for visualization. EGFP fluoresces green when excited by light of 488 nm and may be visualized by epifluorescence microscopy using standard fluorescein isothiocyanate filters (Chalfie et al., 1994) under wavelengths that are not deleterious to living tissues. Since EGFP consists of ~200 amin
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