Zen blue software v3

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.

Caco-2 and MA104 cells were cultured on glass coverslips and incubated after infection with RV-SA11 for 6 and 12 h. These cells were then fixed with 4% w/v paraformaldehyde in PBS and processed for the immunofluorescence technique, as mentioned previously [8 (link)]. Cells were incubated with the antibody dilutions specific to RV-VP6 and β-catenin (1:100) at 4 °C overnight, followed by incubation with FITC- (green) and Rhodamine- (red) conjugated secondary antibodies, respectively, in the dark for 2 h in a 37 °C incubator. The nuclei in the cells were stained with Vectashield containing 4′,6′-diamidino-2-phenylindole (DAPI-blue) and visualized with the help of the Axioplan confocal microscope (Carl Zeiss, Jena, Germany). The ZEN Blue software v3.1 (Carl Zeiss Microscopy, Jena, Germany) was used to process the microscopic images stored in the RGB format. In order to compare different images appropriately, these images were captured under identical excitation and detection settings in one session.

Immunofluorescence images were acquired with a Leica SP5 up‐right confocal microscope ×5, ×10, ×20, ×40, and ×63 objectives and analyzed with ImageJ software and Definiens developer XD 2.5. Immunohistochemistry images were captured with the Zen Blue Software v3.1 (Zeiss), and whole slides were acquired with a slide scanner (AxioScan Z1, Zeiss). For histological quantification of brain metastases at endpoint (5 weeks after intracardiac inoculation of cancer cells), only lesions showing solid and compact distribution of cancer cells were considered as established metastases.