Laser Capture Microdissection of Murine Embryonic Neural Crest Cells
The purpose of this chapter is to provide a step-by-step protocol to enable performance of laser capture microdissection (LCM) on tissue sections from mammalian embryos or postnatal organism stages in order to collect pure populations of neural crest cells from which sufficient amounts of nucleic acids and/or protein can be obtained for quantitative analysis. The methods (1) define a strategy to genetically and indelibly label mammalian neural crest-derived cells with a fluorescent marker, thus enabling their isolation throughout the pre- and postnatal life span of the organism, and (2) describe subsequent isolation by LCM of the labeled neural crest cells, or their derivatives, from embryonic/postnatal tissue cryosections. Details are provided for using the Arcturus PixCell®IIe Laser Capture Microdissection System (Arcturus) and CapSure LCM Caps (Thermo Fisher Scientific), to which the selected cells adhere upon laser-mediated capture. The protocol outlined herein can be applied in any situation wherein limited cellular samples are available for isolation by LCM. Nucleic acids or proteins can be extracted from LCM-isolated cells and processed for high-density gene expression profiling analyses (microarrays or RNA sequencing), Real-Time PCR (q-PCR) for specific candidate gene expression, investigation of DNA methylation, as well as for varied protein analyses.
Key wordsNeural crest cells GFP Laser capture microdissection Embryonic
This report, and any data presented, was supported in part by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103453, and PHS research grants HD053509 and DE018215.
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