Immunofluorescence Imaging of RV-NSP5 in MA104 Cells

MA104 cells, grown on glass coverslips (18 mm square, no. 1; Blue Star) and transfected and/or infected with pmR-ZsGreen1-pre-miR-29b and RV, were fixed with paraformaldehyde (4% w/v in PBS) and further processed as described earlier (Mukhopadhyay et al., 2019 (link)). Cells were incubated with RV-NSP5 (1:200; Rabbit monoclonal; a kind gift from Prof. Koki Taniguchi) diluted in blocking solution at 4°C. After overnight incubation, the cells were washed and further treated with Rhodamine-conjugated goat-anti-mouse (ThermoFisher Scientific: 31660) secondary antibodies for 2 h in the dark in a humidified 37°C incubator. Nuclei were visualized after incubation with Vectashield containing 4′,6′-diamidino-2-phenylindole (DAPI) and mounted on microscope slides. Mounted slides were examined under a Zeiss Axioplan confocal microscope (63x oil immersion). The images were captured and processed using ZEN Blue software v3.1 (Carl Zeiss Microscopy GmbH, Jena, Germany) and saved as 24-bit tagged JPG images in RGB-format. For comparison between different samples, images were collected during a single session at identical excitation and detection settings.