Covalent binding of formalin fixed paraffin embedded brain tissue sections to glass slides suitable for in situ hybridization.
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.
D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D001921:Brain / Q000382:microbiology*; D004279:DNA, Viral / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D007577:JC Virus / Q000235:genetics*; D007968:Leukoencephalopathy, Progressive Multifocal / Q000382:microbiology; D008459:Measles virus / Q000235:genetics; D009693:Nucleic Acid Hybridization; D011120:Polyomavirus / Q000235:genetics*; D011437:Propylamines; D012367:RNA, Viral / Q000032:analysis*; D012821:Silanes; D013539:Simian virus 40 / Q000235:genetics; D014709:Vero Cells
D004279:DNA, Viral; D011437:Propylamines; D012367:RNA, Viral; D012821:Silanes; C477625:amino-propyl-triethoxysilane