Cellular localization of mRNA and protein: in situ hybridization histochemistry and in situ ligand binding.
Powerful methods for the detection of mRNA and proteins in cells and tissue sections have been developed since the mid-1980s. This chapter discusses the applications of in situ hybridization histochemistry and in situ ligand binding to cells in culture and tissue sections. In situ hybridization takes advantage of paired nucleotide interactions between a labeled probe (antisense strand) and the endogenous mRNA (sense strand). Following processing, the mRNA is localized through detection of the disintegration pattern of the radiolabeled probe. Protein-protein interaction is detected in a similar fashion. Proteins are radiolabeled and incubated with tissues that contain target-binding proteins or receptors. On processing, the interaction sites are localized through detection of the radiolabeled probe. The methods are rapid, sensitive, specific, and provide important information regarding the sites of mRNA synthesis, abundance of protein, and the ability of the ligand to interact with the receptor in restricted cellular populations. Application of these techniques to cells in culture allows in vitro manipulation of endogenous mRNA or protein with various hormones or growth factors and a method to detect the results.
Woodruff
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Northwestern University, Departments of Medicine and Neurobiology and Physiology, Chicago, Illinois 60611, USA.
D000818:Animals; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008024:Ligands; D011506:Proteins / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*
D008024:Ligands; D011506:Proteins; D012333:RNA, Messenger