RNA-RNA in Situ Hybridization Using Digoxigenin-Labeled Probes: The Use of High-Molecular-Weight Polyvinyl Alcohol in the Alkaline Phosphatase Indoxyl-Nitroblue Tetrazolium Reaction

A nonradioactive RNA-RNA in situ hybridization method using digoxigenin-labeled probes using indoxyl-nitro blue tetrazolium alkaline phosphatase reaction achieves a 20-fold increase in sensitivity without increasing the background.

Abstract

A nonradioactive RNA-RNA in situ hybridization method using digoxigenin-labeled probes is described. The visualization of hybrids is done using the indoxyl-nitro blue tetrazolium alkaline phosphatase reaction. The addition of polyvinyl alcohols of high molecular weight (40-100 kDa) to the reaction medium enhances the alkaline phosphatase reaction and prevents diffusion of reaction intermediates, resulting in a 20-fold increase in sensitivity without increasing the background. Due to the more localized precipitation of the formazan, the site of hybridization can be determined more precisely.