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        Laser Capture Microdissection (LCM)

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        1022

        This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.

        Theory of LCM
        LCM utilizes an infrared laser integrated into a standard microscope. A transparent cap is attached to a thermoplastic transparent membrane which lies directly on the surface of a routinely prepared tissue section on a glass slide. The investigator examines the tissue section microscopically and activates the laser when the desired cells underlie the target. This in turn activates the membrane with subsequent binding and procurement of the cells of interest (see Figures 1 A and B below). The laser pulse is very brief (approximately 5 msec) and the membrane is activated at 90°C, thus the tissue experiences only a very brief thermal transient as the heat generated from the membrane is rapidly dissipated.

        LCM allows for visualization and image capturing of tissue as it is microdissected, including images of the tissue before and after microdissection. This is critical in maintaining an accurate record of each dissection and correlating histopathology with subsequent molecular results.

        Method
        Once the tissue has been properly processed, sectioned and stained, it is ready for microdissection. The tissue is visualized under the microscope and an initial road map image as well as pre-dissection, post-dissection, and cap images are taken to document the histology, the steps of microdissection, and the microdissected cells, respectively. The laser beam size may be adjusted from 7.5 µm to 30 µm to allow microdissection of either groups of cells or single cells depending on the needs of the investigator.

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        The total number of cells procured depends upon the molecular analytical technique being employed. After the microdissection is complete, the cap with microdissected cells is place onto an Eppendorf tube containing the appropriate buffer for molecular analysis. The tube is then inverted to allow the lysis of the cells and the sample is ready for molecular analysis.

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