Laser-Assisted Microdissection of Membrane-Mounted Sections Following Immunohistochemistry and In Situ

Laser microbeam microdissection (LMM) is an increasingly important histological technique for obtaining homogeneous cell populations and tissue components in order to analyze target-specific changes in genes, gene expression, and proteins. The quality of data obtained with LMM is heavily dependent on the precision with which the target for microdissection can be identified. Since no cover slip is used during LMM, tissue mor-phology is poor compared with traditional light microscopy. This hampers morphologi-cal recognition of targets for microdissection in routinely stained sections and can be a limiting factor in the use of this technique. Immunohistochemistry (IHC) and in situ hybridization (ISH) can improve the identification of specific cell populations in situ in tissue sections, but there are a number of problems in applying these methods to slides prepared for LMM. In this chapter, we present optimized protocols that allow IHC to be performed for detecting a wide range of antigens in conjunction with LMM, both on formalin-fixed paraffin-embedded and on frozen sections. In addition, we present a quick, versatile protocol for performing ISH on archival material suitable for LMM. Key Words: Laser-assisted microdissection; membrane-mounted tissue; MOMeNT; frozen tissue; formalin-fixed paraffin-embedded tissue; immunohistochemistry; in situ hybridization. 1