Axioimager m1 system

The metaphase chromosomes were prepared from infected T cells on day 14 post-infection (dpi) and analyzed for the presence of the MDV genome by FISH [24 (link),31 (link)]. Briefly, MDV genomes were detected using a set of PCR-based MDV probes that were generated using the Biotin PCR Labeling Kit (PromoCell, Heidelberg, Germany) (for primers, see Table 1). Virus genomes were visualized using Cy3 Streptavidin (1:1000; GE Healthcare, PA43001; Munich, Germany), metaphase FISH images were taken using an Axio Imager M1 system and the AxioVision software (Carl Zeiss, Inc.; Oberkochen, Germany) and analyzed with ImageJ (https://imagej.nih.gov/ij/, accessed on 28 November 2021). Appropriate positive and negative controls were included (Figure S2).

Immunofluorescence staining was performed on cultured cells. Cells were fixed in 4% PFA for 20 min and washed with PBS/0.01% Triton X-100 (PBST) and permeabilized with 0.2% Triton X-100 in PBS at room temperature for 5 min. Next, blocking was performed for 1 h using 5% normal goat serum in PBS. Following this step, the cells were stained for the HA-tag (1:1,000, Cell Signaling) diluted in PBST/1% normal goat serum at 4°C overnight. The next day, cells were washed with PBST, and incubated with goat anti-rabbit or goat anti-mouse 594 secondary antibodies (all at 1/500, Invitrogen) in PBST at room temperature for 2 h. Following this incubation, cells were washed with PBST three times for 10 min each and mounted in Vectashield mounting media with DAPI (Vector Labs, Burlingame, CA) and cover slipped for imaging. Fluorescence microscopy was performed using a Zeiss AxioImager M1 system equipped with epifluorescence filters, a Zeiss monochrome digital camera and AxioVision software. All images were processed in Adobe Photoshop for brightness/contrast, orientation and background correction to better illustrate staining patterns.