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Laser Capture Microdissection of Cells Labeled with Enhanced Green Fluorescent Protein

o acid residues, it may be expressed either as a tag at the c-terminus of an endogenous protein (a fusion protein) or as a free peptide in the cytoplasm. Owing to the high solubility of EGFP when expressed as a free protein, fixation is required to prevent leaching of the protein and retain fluorescence after cryosectioning. Recently, chromosomal expression of EGFP from transgenic reporter constructs has been adopted for use as a cell type-specific label in transgenic mice. The combination of cell type-specific EGFP expression with the powerful technique of LCM allows the isolation of RNA from cell types, which are otherwise morphologically similar to surrounding cells or which cannot be detected by immunolabeling. This method describes the isolation of an embryonic stem cell population, the neural crest, that has been genetically labeled with EGFP and the subsequent extraction of mRNA and amplification of neural crest cell gene markers (Bhattacherjee et al., 2004).


Equipment and Reagents
This protocol requires the following reagents:
◊ Phosphate buffered saline (PBS)
◊ 4% Paraformaldehyde (PFA) dissolved in PBS
◊ Silane Prep Slides or equivalent (Sigma Aldrich, St. Louis, MO, Cat. # S-4651)
◊ CapSure HS LCM Caps (Arcturus, Cat. # LCM 0204)
◊ Optimal cutting temperature compound (OCT - Tissue-Tek, Cat. # 4583)
◊ PicoPure RNA isolation kit (Arcturus, Cat. # KIT0204)
◊ RiboAmp RNA amplification kit (Arcturus, Cat. # KIT0203)
◊ Superscript II reverse transcriptase (Invitrogen Life Technologies, Inc., Carlsbad, CA, Cat. # 18064-022)


Equipment and Labware
The following laboratory equipment is required:
◊ PixCell IIe LCM Instrument equipped with Fluor 300 epifluorescence optics optimized for EGFP
◊ TaqMan ABI Prism 7000 S
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