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

Applied Immunohistochemistry & Molecular Morphology : AIMM
Hiroaki NittaThomas M Grogan

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...Continue Reading

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Citations

May 6, 2005·Journal of Dermatological Science·Tsutomu SomaJiro Kishimoto
Feb 9, 2005·Kidney International·Bassam AlchiFumitake Gejyo
Sep 28, 2012·Journal of Laboratory Automation·David SabourinMartin Dufva
Feb 17, 2006·Experimental Dermatology·A TakaokaS Nakaike
Sep 26, 2006·Journal of Dermatological Science·Yuji ShirakataKoji Hashimoto
Aug 19, 2007·American Journal of Physiology. Regulatory, Integrative and Comparative Physiology·Hiroko NishimuraTadashi Yamamoto
Sep 9, 2005·The Biochemical Journal·Michiko MitsuishiMakoto Murakami
Apr 7, 2007·Developmental Biology·Roland E KälinAndré W Brändli
Apr 13, 2007·The Journal of Investigative Dermatology·Ritsuko EhamaJiro Kishimoto
Jan 13, 2009·General and Comparative Endocrinology·Keith K LauHiroko Nishimura

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