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-embedding in situ hybridization (ISH) strategies. One unique resin that has not been fully investigated or exploited with respect to light microscopic ISH is a methacrylate mixture. The advantage of this resin is its ability to be removed from tissues postsectioning, dramatically increasing 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 protocols are described, using both end-labeled oligonucleotides and randomly primed DNA probes (200-400 BPS), 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 collagen transcripts in foot tissue of two marine bivalves. MEADE ISH has proved extremely versatile and will likely be suitable for many future applications.
Warren
K C
KC
Graduate College of Marine Studies, University of Delaware, Lewes, Delaware 19958, USA.
Coyne
K J
KJ
Waite
J H
JH
Cary
S C
SC
D000818:Animals; D001419:Bacteria / Q000302:isolation & purification; D049872:Bivalvia / Q000382:microbiology; D005880:Gills / Q000382:microbiology; D017403:In Situ Hybridization / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008689:Methacrylates; D020366:Methylmethacrylate; D008768:Methylmethacrylates; D008974:Mollusca / Q000382:microbiology; D015345:Oligonucleotide Probes; D011347:Procollagen / Q000032:analysis; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D013559:Symbiosis; D016610:Tissue Embedding
D008689:Methacrylates; D008768:Methylmethacrylates; D015345:Oligonucleotide Probes; D011347:Procollagen; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D020366:Methylmethacrylate; C016284:butyl methacrylate