Covalent binding of formalin fixed paraffin embedded brain tissue sections to glass slides suitable for in situ hybridization |
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| Authors: | W W Tourtellotte A N Verity P Schmid S Martinez P Shapshak |
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| Affiliation: | 1. Neurology and Research Services, VAMC West Los Angeles, Wadsworth Division, Los AngelesUSA;2. Department of Pediatrics, Harbor UCLA Medical Center, Los Angeles, California, U.S.A.;3. Department of Neurology, Reed Neurological Research Center, UCLA School of Medicine, Los AngelesU.S.A.;1. Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal;2. Department of Chemistry & QOPNA, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal;3. cE3c- Centre for Ecology, Evolution and Environmental Changes/Azorean Biodiversity Group & University of Azores, Rua Mãe de Deus, 9501-801 Ponta Delgada, Portugal;1. Johns Hopkins Drug Discovery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;2. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;3. IRCCS Fondazione Don Carlo Gnocchi ONLUS, Laboratory of Nanomedicine and Clinical Biophotonics (LABION), Milan, Italy;4. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;5. Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;6. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;7. Department of Psychiatry and Behavioral Science, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;8. Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;1. Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany;2. German Centre for Integrative Biodiversity Research (iDiv), Deutscher Platz 5e, 04103 Leipzig, Germany;3. Friedrich Schiller University Jena, Institute for Ecology, Jena, Germany;4. Molecular Interaction Ecology, Institute of Water and Wetland Research (IWWR), Radboud University, PO Box 9010, 6500 GL Nijmegen, The Netherlands;5. Research Group Sequestration and Detoxification in Insects, Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany;6. The Helmholtz Centre for Environmental Research (UFZ), Permoserstraße 15, 04318 Leipzig, Germany;7. Martin Luther University Halle-Wittenberg, Universitätsplatz 10, 06108 Halle, Germany |
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| Abstract: | ![]()
A novel method for covalently binding formalin fixed paraffin embedded (FFPE) tissue sections to glass microscope slides is validated suitable for in situ hybridization (ISH). Using the organosilane methodology of Maples (1985), 100% tissue adhesion is reported with no nonspecific probe binding, staining, or autoradiographic artefacts. JC viral nucleic acid sequences are successfully detected in FFPE progressive multifocal leukoencephalopathy brain tissue and the Tm of the hybridized product is estimated. From the Tm the most stringent washing condition resulting in an optimal signal to noise ratio is determined. A comparison is made between currently used methods of tissue adhesion and the proposed organosilane methodology. This methodology greatly facilitates studies of conditions for ISH and elucidation of mechanisms of viral infections requiring consecutive FFPE sections. It is also applicable to studies using cryosections and cultured cells. |
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