Chemical fixation, immunofluorescence, and immunogold labeling of electron microscopical sections

Abstract

Knowledge about the spatiotemporal distribution patterns of proteins and other molecules of the cell is essential for understanding their function. A widely used technique is immunolabeling which uses specific antibodies to reveal the distribution of molecular components at various structural levels. Immunofluorescence gives an overview about the distribution of molecules at the level of the fluorescence or confocal laser scanning microscope. Electron microscopy offers the highest resolution of morphological techniques and is thus an indispensable tool for the analysis of molecule distribution patterns at the subcellular level. In this chapter we describe selected routine methods for immunofluorescence and for labeling ultrathin sections of resin-embedded material with antibodies conjugated to colloidal gold, including protocols for chemical fixation, embedding, and sectioning.