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Working With Laser Capture Microdissection Samples


Laser Capture Microdissection (LCM) is a method for obtaining pure populations of cells from heterogeneous samples. LCM permits the selection and capture of cells, cell aggregates, and discrete morphological structures from thin tissue sections. For LCM, frozen and OCT embedded, or formalin-fixed and paraffin embedded tissue sections are processed, stained, and dehydrated, then scanned under an inverted microscope. Cells are visualized through a thermoplastic film which is attached to the bottom of an optically clear microfuge tube cap. A laser pulse, directed onto the target cells through the film, melts the film and allows it to flow onto the targeted area where it cools and bonds with the underlying cell(s). The film including the adhered cells or clusters is then lifted. Captured cells can then be used for nucleic acid studies, including SNP analysis, endpoint and real-time RT-PCR, and mRNA expression profiling.


Formalin vs. Frozen Sections
LCM is usually carried out on frozen sections when samples will be used for RNA isolation. This is because formalin fixation introduces crosslinks between nucleic acids and proteins, resulting in fragmentation of the RNA. Freezing avoids crosslinking but introduces a different problem. Endogenous nucleases are not irreversibly inactivated, and can become active during the tissue processing steps (fixation, staining, destaining, and dehydration) carried out after sectioning.


An Improved Protocol for Staining
Ambion scientists performed a detailed analysi
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