Immunoguided microdissection techniques

Abstract

Over the past 15 years, laser-based microdissection has improved the precision by which scientists can procure cells of interest from a heterogeneous tissue section. However, for studies that require a large amount of material (e.g., proteomics) or for cells that are scattered and difficult to identify by standard histological stains, an immunostain-based, automated approach becomes essential. In this chapter, we discuss the use of immunohistochemistry (IHC) and immunofluorescence (IF) to guide the microdissection process via manual and software-driven auto-dissection methods. Although technical challenges still exist with these innovative approaches, we present here methods and protocols to successfully perform immuno-based microdissection on commercially available laser dissection systems.

Fig. 1

Schematic representation of immunoguided microdissection techniques. Initial immunostaining step represented by the antibody complex attached to a labeling enzyme (circles).

Fig. 2

AutoScanXT software guided dissection of a cytokeratin AE1/AE3 IHC-stained prostate tissue. (a) Screen capture of the static jpeg image where ROIs (circles) and background (squares) as determined by the user. Inset highlights the symbols used to select the ROIs and background. The symbols are enhanced in the inset for visualization purposes. (b) Screen capture of the ROIs (circled areas) selected by the AutoScanXT analysis module. (c) Image of the LCM cap showing the dissected epithelial cells. (d) Image of the remaining tissue following AutoScanXT-directed dissection. All images captured at 100× magnification.