Qualitative and quantitative detection of alkaline phosphatase coupled to an oligonucleotide probe for somatostatin mRNA after in situ hybridization using unfixed rat brain tissue

Abstract

In situ hybridization (ISH) of somatostatin (SOM) mRNA was carried out on sections of rat brain using an alkaline phosphatase (AP) coupled oligonucleotide probe. Different hybridization and AP development conditions were tested for qualitative and quantitative detection of target mRNA on sections of unfixed tissue. Hybridization signal intensities after 24 h of hybridization were high. Comparison with adjacent formaldehyde-fixed tissue sections and hybridization for various lengths of time (2-42 h) indicated that in unfixed tissue retention of SOM mRNA was at least as high as after fixation, and that the mRNA was not degraded during hybridization. The use of tetranitroblue instead of nitroblue tetrazolium chloride in the AP detection medium provided a superior signal-to-noise ratio, and medium stability was improved for quantitative studies on unfixed sections by adding 10% polyvinyl alcohol at pH 8.5. Microphotometric measurements of mean optical densities (MOD) of the formazan reaction product in a defined area within individual neurons of the lateral central amygdaloid nucleus showed a linear increase over the first 23 h of AP reaction time. The mean MOD values per neuron were comparably high in various equally thick sections of the nucleus and increased with section thickness in a linear manner. The findings indicate that the ISH and detection reagents penetrate the entire section and that there is a linear relationship between the amount of AP reaction product measured and the amount of mRNA present in the measured area. Thus, ISH using an AP-coupled oligonucleotide on sections of unfixed tissue appears suitable for quantitative mRNA detection.