USE OF METHACRYLATE DE-EMBEDDING PROTOCOLS FOR IN-SITU HYBRIDIZATION ON SEMITHIN PLASTIC SECTIONS WITH MULTIPLE DETECTION STRATEGIES

New plastic resins are gradually replacing traditional paraffin-embedd ing in situ hybridization (ISH) strategies. One unique resin that has not been fully investigated or exploited with respect to light microsc opic ISH is a methacrylate mixture. The advantage of this resin is its ability to be removed from tissues postsectioning, dramatically incre asing hybridization signal compared to that obtained in other plastics . The goal of this study was to evaluate the general applicability of the methacrylate embedding acetone de-embedding (MEADE) technique for ISH investigations. Several high-resolution, high-sensitivity ISH prot ocols are described, using both end-labeled oligonucleotides and rando mly primed DNA probes (200-400 sps), signal amplification by catalyzed reporter deposition (CARD), and chromogenic and fluorescent detection methods. With slight modification, the MEADE ISH technique permitted localization of bacterial symbionts in fragile gill tissue and collage n transcripts in foot tissue of two marine bivalves. MEADE ISH has pro ved extremely versatile and will likely be suitable for many future ap plications.