In Situ Hybridization
This chapter reviews the basic principles and approaches of In situ hybridization (ISH) and discusses some technical advances, the use of ISH for the diagnosis of specific diseases and its uses in research. Isotopic labeling may be used to detect low copy sequences and multiple nucleotide sequences, or further to combine with immunohistochemistry. Hybridization signals are detected by auto-radiography, using either liquid emulsion or x-ray film and quantified by using silver grain counting or semi-quantified by densitometry. Fluorescent in situ hybridization (FISH) is commonly used in molecular cyto-genetics. It is particularly useful for interphase cyto-genetics where multicolor hybridization requires high resolution. Applications of FISH include karyotype analysis, gene mapping, deoxyribonucleic acid (DNA) replication and DNA recombination. The relative insensitivity of nonisotopic ISH has been cited as an important limiting factor in many applications, especially in diagnostic pathology. Improving the sensitivity of ISH has been a goal of many investigators.