In situ hybridization

This chapter focuses on the use of in situ hybridization for tissue sections with particular emphasis on paraffin-embedded material. Identification of the temporal and spatial patterns of gene expression can provide important clues about gene function. This holds particularly true in embryonic development. In situ hybridization makes it possible to localize transcripts directly in cells within the context of their tissues and organs. In situ hybridization relies on the annealing of labeled nucleic acid probes to DNA or RNA sequences within a tissue section or, in the case of whole mount protocols, within an organ or fragment of tissue. The signal to noise ratio depends on the specificity, length, complexity, and accessibility of the probe to the hybridization substrate. In the case of in situ hybridization, accessibility gains considerable importance, as the tissue section has a thickness that must be penetrated by the probe without compromising histological integrity. The in situ hybridization technique has been adapted effectively for studies in a variety of organisms ranging from Drosophila to humans. This method can be employed using DNA or RNA probes to hybridize to a variety of tissue targets including endogenous genes (DNA), viral genes (DNA or RNA), and endogenous or viral transcripts (RNA).