4] Antifading agents for confocal fluorescence microscopy

Abstract

The chapter discusses strategies to reduce specimen-associated background fluorescence and the properties and performance of several antifading agents during the acquisition of multiple optical sections. Discussions are focused on indirect immunofluorescence, and other in situ localization methods are also discussed. Currently, single- and dual labeling immunofluorescence, imaging of green fluorescent protein, and fluorescent in situ hybridization (FISH) are the most widely used applications of the confocal laser scanning microscope (CLSM). Single- or dual-labeling immunofluorescence may involve either a direct or an indirect method. The greatest advantage of the CLSM is its ability to discriminate between signals originating from in-focus and out-of-focus optical planes to produce digital images that can be merged pixel by pixel to generate perfectly registered two- and three-dimensional renditions. The chapter also discusses specimen preparation, antifading agents, and acquisition of fading fluorescent images. The reduction of specimen-associated background fluorescence is performed immediately after paraformaldehyde fixation of the sample.