Localization of adenovirus DNA by in situ hybridization electron microscopy.
Biotinylated deoxyadenosine triphosphate (dATP) (Bio-7-dATP) and 3H deoxythymidine triphosphate (dTTP) labeled adenovirus DNA were hybridized in situ to thin sections of Lowicryl K4M-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 3H-dTTP labeled probe by electron microscopic autoradiography. On Lowicryl K4M 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; b) superior morphological preservation and spatial resolution; c) precise localization; and d) on Lowicryl K4M sections, signal to noise equivalent to radiolabeling.
Jiao
R
R
Department of Biology, Peking University, Beijing, People's Republic of China.
Yu
W
W
Ding
M
M
Zhai
Z
Z
D000180:Acrylic Resins; D000260:Adenoviruses, Human / Q000235:genetics*; D001345:Autoradiography; D001710:Biotin; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis / Q000235:genetics; D016610:Tissue Embedding
D000180:Acrylic Resins; D015342:DNA Probes; D004279:DNA, Viral; D001710:Biotin; C037883:Lowicryl 4KM resin