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BrUTP incorporation assay.

RT Raquel Tenorio
IC Isabel Fernández de Castro
JK Jonathan J. Knowlton
PZ Paula F. Zamora
CL Christopher H. Lee
BM Bernardo A. Mainou
TD Terence S. Dermody
CR Cristina Risco
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Newly synthesized viral RNA was labeled in reovirus-infected cells using a bromouridine (BrU) incorporation assay. HeLa cells were adsorbed with reovirus at an MOI of 1 PFU/cell. At 14 h postadsorption, cells were incubated for 6 h with 50 µg/ml α-amanitin (Sigma) to block cellular RNA synthesis. At 15 h postinfection, cells were incubated for 5 h with 10 mM BrU (Sigma), washed with PBS, fixed for 1 h with 4% PFA in PBS, and processed for cryosectioning and immunogold labeling using a monoclonal antibromodeoxyuridine antibody (Sigma) diluted 1:50 in saturation buffer for 1 h followed by a secondary antibody conjugated with 10-nm colloidal gold particles for 30 min. Samples were imaged using a JEOL JEM-1011 transmission electron microscope.

Copyright and License information:  ©2018 Tenorio et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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