In Situ Hybridization Protocol for Overall Preservation of mRNA in Fixed Tissues with a Poly d(T) Oligonucleotide Probe

Abstract

Nonisotopic in situ hybridization (ISH) for polyadenylated messenger RNA (mRNA) sequences was performed on a variety of tissues fixed in 3 commonly used fixatives, 100% ethanol, 10% neutral buffered formalin (NBF), and Bouin solution. The method utilized a synthetic 20 base biotin-labeled poly T oligonucleotide probe in combination with capillary action technology and was performed in under 2 hr. Poly A sequences were detected in tissues fixed with all 3 fixatives; however, optimal protocols varied between aldehyde fixed and alcohol fixed tissues. For alcohol fixed tissues, poly A detection was achieved with a protocol that excluded the use of prehybridization protease and acid denaturation. These parameters were necessary in aldehyde tissues for optimal detection of poly A sequences. Poly A sequences were seen in tissues fixed in ethanol or NBF from 2–216 hr. Strong signal was identified in tissues fixed inBouin for less than 8 hr after which signal was decreased or not present, regardless of the method used. ISH with the poly T oligonucleotide may be a rapid way to determine the suitability of a routinely processed block for in situ mRNA hybridization.(The J Histotechnol 16:315, 1993)

Keywords:

• capillary action
• in situ hybridization
• oligonucleotides
• poly A sequences