Improved sensitivity for high resolution in situ hybridization using resinextraction of methyl methacrylate embedded material

An in situ hybridization procedure resulting in both high resolution and se nsitivity was established by using the removable methyl methacrylate resin, Technovit 9100, Young bicellular pollen of tobacco (Nicotiana tabacum L. S R-1) was embedded in Technovit 9100 resin and sectioned, The resin was extr acted with (2-methoxyethyl)-acetate followed by in situ hybridization with cRNA probes to detect cytoplasmic 18S/25S rRNA. Signal intensity obtained b y this procedure was approximately twice as great as that obtained by an ea rlier procedure using Technovit 7100, a glycol methacrylate resin that cann ot be removed from sections. This improvement in sensitivity made it possib le to observe subcellular localization of small amounts of RNA as revealed by visualization of plastid 23S rRNA in a generative cell of Plumbago auric ulata pollen.