A Rapid and Highly Sensitive In Situ mRNA Hybridization Method With Digoxigenin Labeled cRNA Probes

Abstract

To raise detection sensitivity, the rapid in situ hybridization (ISH) method using cRNA probes has been improved. The rapid ISH method with the Micro Probe System and digoxigenin (DIG)-labeled cRNA probes was used to detect insulin and glucagon mRNAs in 10% formalin-fixed and paraffin-embedded rat pancreatic tissues. The reagent used was ready-made and fitted for the Micro Probe System rather than proteinase K solution and the hybridization mixture. A comparative study was made of stainability by adding various reagents to the ready-made hybridization mixture (Brigati Probe Diluent). Detection sensitivity for insulin and glucagon mRNAs was significantly increased when 50% deionized formamide and 8% dextran sulfate were added to the hybridization mixture for 2 h at 50°C. Moreover, omitting probe linearization for 3 min at 105°C during the hybridization step allowed plainer stainability and less tissue damage. When the nonspecific reaction was strong, it was possible to solve the problem by processing the section in 2% normal swine serum just before the antigen- antibody reaction. Using this modified method, we were able to detect insulin and glucagon mRNAs clearly within three hours. The rapid ISH with cRNA probes described here could have many applications in various research fields and clinical laboratories. (The J Histotechnol 29:91, 2006)Submitted November 14, 2005 accepted February 12, 2006.