Immunofluorescence and FISH

This chapter presents and summarizes two very important techniques in biomedical research, immunofluorescence and fluorescence in situ hybridization (FISH), which relay completely in the use of fluorescent methods to achieve their results. Although the particular molecules involved are different both methods make use of the same approach: the detection of a specific molecular sequence or moiety by a specific molecule (antibody or nucleic acid sequence) that has a fluorophore attached to it. This fluorophore permits the detection of the specific target. An improvement over fluorophore-tagged antibodies has been the introduction of fluorescent proteins, generically known as GFPs (green fluorescent proteins) or FPs (fluorescent proteins). Here we will elaborate a little bit further on their practical applications, as one can think of FPs as a “subtype” of immunofluorescence. Strictly speaking immunofluorescence and FISH provide no new concept to the principles of fluorescence. They rely on using a more or less classical fluorophore and the real breakthrough was the use of the exquisite specificity of antibodies and nucleobase complementarity to recognize certain substrates. However, immunofluorescence represents probably the most widespread employed technique in fluorescence microscopy. And FISH is only a step behind if at all in regards as its use as a diagnostic and research tool in genetics and medicine. Therefore we will introduce the basics of both techniques and some of their most promising recent applications in this chapter.

Keywords: Anti-fading agent, Antibody, Antigen, Chromobody, Chromosome painting (M-FISH), Comet-FISH, Counterstaining dye, Direct immunofluorescence, Enzyme-linked immunosorbent assay (ELISA), Fluorescence in situ hybridization (FISH), Fluorescent protein, Fluorophore-tagged antibody, Genetic analysis, Immunofluorescence, Indirect immunofluorescence, Nanobody, Western blot.