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Localization of adenovirus DNA by in situ hybridization electron microscopy
Author(s) -
Jiao Renjie,
Yu Wendou,
Ding Mingxiao,
Zhai Zhonghe
Publication year - 1992
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.1070210104
Subject(s) - biotinylation , in situ hybridization , hybridization probe , dna , microbiology and biotechnology , in situ , biology , biotin , cell nucleus , electron microscope , chemistry , biophysics , nucleus , biochemistry , physics , optics , messenger rna , organic chemistry , gene
Biotinylated deoxyadenosine triphosphate (dATP) (Bio‐7‐dATP) and 3 H deoxythymidine triphosphate (dTTP) labeled adenovirus DNA were hybridized in situ to thin sections of Lowicryl K 4 M‐embedded and whole‐mount extracted HeLa cells infected with adenovirus. The biotinylated probe was detected by exposing the extracted cells or sections to antibodies against biotin followed by colloidal gold‐conjugated secondary antibodies and then critical‐point dried while 3 H‐dTTP labeled probe by electron microscopic autoradiography. On Lowicryl K 4 M sections, gold particles and silver grains were mainly restricted in the nucleus. Furthermore, whole‐mount results suggested that replicating adenovirus DNA is localized on the nuclear matrix of its host cell. In this paper, the described non‐radioactive procedures for hybrid detection offered several advantages: (a) rapid signal detection; (ob) superior morphological preservation and spatial resolution; (c) precise localization; and (d) on Lowicryl K 4 M sections, signal to noise equivalent to radiolabeling.