Laser-assisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis - Identification of cell populations using immunohistochemistry and in situ hybridization

Laser microbeam microdissection (LMM) is an increasingly important method f or obtaining pure cell samples for genetic and proteomic analysis. Immunohi stochemistry (IHC) and in situ hybridization (ISH) are useful techniques fo r targeting specific cell populations for microdissection but are difficult to apply with the tissue support membranes often used during LMM. Using de tection of cytokeratins and Epstein-Barr virus gene products in head and ne ck carcinoma as a model, we describe optimized protocols for membrane and s ection preparation and for low temperature antigen retrieval that allow IHC and ISH to be used reliably on membrane mounted paraffin tissue sections. Visualization of cellular targets was markedly improved by staining and thi s could be further improved using a variety of optical media before microdi ssection. Tissue fragments thus stained were suitable for subsequent polyme rase chain reaction analysis of extracted DNA using standard techniques. Th ese IRC and ISH procedures are generally applicable and will be useful for detecting a wide range of antigens and nucleic acids in paraffin sections i n conjunction with LMM.