Click it edu reaction additive

Primary HMVEC-d and HFF cells seeded on glass chamber slides (Nalgene Nunc International) were uninfected, KSHV infected (30 DNA copies/cell), or HSV-1 infected (1pfu/cell), fixed for 15 min with 4% paraformaldehyde, and permeabilized using 0.2% Triton X-100 for 5 min. Cells were then washed and blocked using Image-iT signal enhancer (Life Technologies) for ~20 min followed by incubation with primary antibodies and then incubated with secondary antibodies conjugated with fluorescent dye. To detect EdU labeled viral genome, cells were fixed, permeabilized and blocked with Image-iT signal enhancer for 20 min. A CLICK reaction was performed for 30 min at RT using Click-iT EdU reaction additive (Life Technologies), copper sulphate, EdU reaction buffer and Alexa Fluor 594 azide. Cells were observed by Nikon Eclipse 80i microscope, and analyzed with Metamorph digital imaging software. All images were acquired at 40X magnification.

HMVEC-d cells were seeded on glass 8-well chamber slides (Nalgene Nunc International, Naperville, IL) and uninfected or KSHV infected (30 DNA copies/cell or latently infected) cells were fixed for 15 min with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100 for 5 min. Cells were then washed and blocked with Image-iT FX signal enhancer (Life Technologies) for 20 min. The cells were reacted with primary antibodies against the specific proteins, followed by fluorescent dye-conjugated secondary antibodies. To detect EdU labeled viral genome, cells were fixed, permeabilized and blocked with Image-iT FX signal enhancer (Life Technologies) for 20 min. A CLICK reaction was performed for 30 min at RT using Click-iT EdU reaction additive (Life Technologies), EdU reaction buffer, copper sulphate and Alexa Fluor 594 azide. Cells were observed by Nikon Eclipse 80i microscope, and analyzed with Metamorph digital imaging software. All experiments were performed three independent times and three different fields with a minimum of 20 cells were analyzed. All images were acquired at 40 X magnification.

Mouse BMDMs were seeded on glass chamber slides (ThermoFisher Scientific) infected with or without HSV-1 (MOI, 10). Cells were then washed and blocked using Image-iT signal enhancer (Life Technologies) for 20 min, followed by incubation with primary antibody (Invitrogen, PA5-86634), and then incubated with secondary antibodies conjugated with fluorescent dye. To detect EdU labeled viral genome, cells were fixed, permeabilized, and blocked with Image-iT signal enhancer for 20 min. A CLICK reaction was performed for 30 min at RT using Click-iT EdU reaction additive (Life Technologies), copper sulfate, EdU reaction buffer, and Alexa Fluor 555 azide (ThermoFisher Scientific). To detect EU-labeled IAV, a CLICK reaction was performed using Click-iT EU reaction additive (Life Technologies), copper sulfate, EU reaction buffer, and Alexa Fluor 488 azide according to the manufacturer’s instructions (ThermoFisher Scientific). Cells were observed by Olympus FV100MPE microscope, and analyzed with FV10-ASW_Viewer imaging software.