QUANTITATIVE COMPARISON OF PRETREATMENT REGIMENS USED TO SENSITIZE IN-SITU HYBRIDIZATION USING OLIGONUCLEOTIDE PROBES ON PARAFFIN-EMBEDDED BRAIN-TISSUE

Paraffin embedding of tissue is generally perceived to dramatically re duce RNA detectability. As a consequence, in situ hybridization on par affin-embedded tissue is largely confined to detection of high-copy RN A species (e.g., viral RNA) and/or to detection using typically more s ensitive cDNA probes or riboprobes. In this study, several procedures for in situ hybridization on paraffin-embedded rat tissue using oligon ucleotide probes complementary to cellular transcripts were developed and quantitatively compared. Certain pretreatments showed marked incre ases in sensitivity compared to untreated sections. Furthermore, throu gh quantitative assessment using image analysis, sensitivity of optima l pretreatments was equal to that of routinely used fresh-frozen, post fixed tissue sections. The development of such techniques permitting i n situ hybridization to be carried out on paraffin-embedded tissue all ows a comparison of protein and mRNA distribution to be made in adjace nt sections and provides the potential for double labeling by in situ hybridization and immunohistochemistry which may not be possible on po st-fixed frozen sections.