IN-SITU HYBRIDIZATION PROTOCOL FOR OVERALL PRESERVATION OF MESSENGER-RNA IN FIXED TISSUES WITH A POLY D(T) OLIGONUCLEOTIDE PROBE

Nonisotopic in situ hybridization (ISH) for polyadenylated messenger R NA (mRNA) sequences was performed on a variety of tissues fixed in 3 c ommonly used fixatives, 100% ethanol, 10% neutral buffered formalin (N BF), and Bouin solution. The method utilized a synthetic 20 base bioti n-labeled poly T oligonucleotide probe in combination with capillary a ction technology and was performed in under 2 hr. Poly A sequences wer e detected in tissues fixed with all 3 fixatives; however, optimal pro tocols varied between aldehyde fixed and alcohol fixed tissues. For al cohol fixed tissues, poly A detection was achieved with a protocol tha t excluded the use of prehybridization protease and acid denaturation. These parameters were necessary in aldehyde tissues for optimal detec tion 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 tissu es fixed in Bouin for less than 8 hr after which signal was decreased or not present, regardless of the method used. ISH with the poly T oli gonucleotide may be a rapid way to determine the suitability of a rout inely processed block for in situ mRNA hybridization.