Retrieval of cellular mRNA in paraffin-embedded human brain using hydrated autoclaving

The aims of this study were to determine the optimal pretreatment of paraffin-embedded human brain sections for in situ hybridization using oligonucleotide probes. A selection of heating and enzymatic methods were compared and their effect on tissue morphology in addition to their ability to sensitise hybridization signal was investigated. In situ hybridization was carried out using a [35S]dATP 3′-end-labeled 30 base oligonucleotide specific for the human N-methyl-d-aspartate (NMDA) NR1-1 receptor subunit. In human hippocampus, NMDA NR1-1 mRNA was detected in the dentate gyrus, CA1, CA2 and CA3 pyramidal neurons and subiculum. The optimal pretreatment of paraffin-embedded sections was autoclaving in citrate buffer, pH 6.0. This novel technique was as sensitive as carrying out in situ hybridization on routinely used fresh-frozen, post-fixed sections, but offers significant advantages including preservation of superior morphology, more efficient, safe and stable storage dynamics and ability to conduct in situ hybridization and immunohistochemical detection methods on adjacent or identical sections.