title: Application of automated mRNA in situ hybridization for formalin-fixed, paraffin-embedded mouse skin sections: effects of heat and enzyme pretreatment on mRNA signal detection.
abstract: Recently, an automated mRNA in situ hybridization application was introduced for the Ventana Discovery instrument. The application was designed so that all necessary steps from baking through signal detection were completed within 1 day on the instrument. We applied this technology for visualizing the expression site of versican in formalin-fixed mouse skin paraffin tissue sections. Our focus of this study was to demonstrate the effects of protease digestion or heating pretreatment, termed cell conditioning, on the hybridization signal using a well characterized versican antisense riboprobe. Paraffin sections were automatically deparaffinized, fixed, and acid-treated. Then, the tissue sections were subjected to protease digestion alone (3 strengths), cell conditioning alone, or the combination of cell conditioning and protease digestion. Hybridization was performed with digoxigenin-labeled versican antisense probe (20 ng/slide) for 6 hours, and the signal was detected using a Nitro blue Tetrazolium chloride 5-Bromo-4-cloro-3-indolyl phosphate toluidine salt (NBT/BCLIP) substrate solution for 3 hours on the instrument. Cell conditioning alone did not produce any signal, whereas the highest strength of protease digestion produced noticeable background staining. However, when cell conditioning and mild protease digestion were combined, the signal for versican mRNA was clearly demonstrated in the hair papilla region. Thus, we demonstrated the effects of the cell conditioning step followed by mild protease digestion for enhancing the mRNA target staining compared with protease digestion or the cell conditioning step alone. We verified that the automated in situ hybridization process was applicable for formalin-fixed mouse skin paraffin sections and that the automated 1-day protocol is simple and reproducible. The precise control of automation allows fine tuning of temperature and enzyme dose to find the optimized assay condition for the signal to noise ratio and morphology.
authors.affiliation: Ventana Medical Systems, Inc, Tucson, Arizona 85737, USA. hnitta@ventanamed.com
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nitta
authors.forename: Jiro
authors.initials: J
authors.lastname: Kishimoto
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Grogan
mesh_terms: D000818:Animals; D001331:Automation; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D005557:Formaldehyde; D006358:Hot Temperature; D017403:In Situ Hybridization / Q000379:methods*; D037181:Lectins, C-Type; D051379:Mice; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000032:analysis*; D012867:Skin / Q000166:cytology*; D016707:Tissue Fixation; D053675:Versicans
chemical_list: D011508:Chondroitin Sulfate Proteoglycans; D037181:Lectins, C-Type; D012333:RNA, Messenger; C508323:Vcan protein, mouse; D053675:Versicans; D005557:Formaldehyde; D010450:Endopeptidases