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 reduce RNA detectability. As a consequence, in situ hybridization on paraffin-embedded tissue is largely confined to detection of high-copy RNA species (e.g., viral RNA) and/or to detection using typically more sensitive cDNA probes or riboprobes. In this study, several procedures for in situ hybridization on paraffin-embedded rat tissue using oligonucleotide probes complementary to cellular transcripts were developed and quantitatively compared. Certain pretreatments showed marked increases in sensitivity compared to untreated sections. Furthermore, through quantitative assessment using image analysis, sensitivity of optimal pretreatments was equal to that of routinely used fresh-frozen, postfixed tissue sections. The development of such techniques permitting in situ hybridization to be carried out on paraffin-embedded tissue allows a comparison of protein and mRNA distribution to be made in adjacent sections and provides the potential for double labeling by in situ hybridization and immunohistochemistry which may not be possible on post-fixed frozen sections.