In situ Hybridization

Hybridization is the formation of hybrid nucleic acid molecules with complementary nucleotide sequences in DNA:DNA, DNA:RNA, or RNA:RNA forms. In situ hybridization is a highly sensitive technique that allows detection and localization of specific DNA or RNA molecules in morphologically preserved isolated cells, histological tissue sections, or chromosome preparations. In situ hybridization has broad range of applications and has been used to (a) localize viral infection, (b) identify sites of gene expression, (c) analyze mRNA transcription and tissue distribution, and (d) map gene sequences in chromosomes. There are several advantages of the use of in situ hybridization including the fact that it can be applied to archival materials and frozen tissues and can be combined with immunohistochemistry to detect protein as well as mRNA of interest or phenotype of cells expressing the target genome, detecting more than one nucleic acid sequences using different labeling methods.The major steps involved in in situ hybridization are as follows: probe preparation and labeling, tissue fixation, permeabilization, hybridization, and signal detection and these are described in detail in this chapter.