Zen 2012 blue edition

The culture plates were inserted into an on-stage incubator on a Zeiss LSM780 confocal microscope at 37°C and 5% CO₂. The time-lapse images were captured at 5-20 min intervals and processed with ZEN 2012 (blue edition) (Zeiss), Huygens Professional (Scientific Volume Imaging) and Fiji (Schindelin et al., 2012 (link)).

For immuno-fluorescent staining, chicken 2D enteroids were grown on 2% Matrigel (Corning) coated transwell inserts (VWR, 0.33 cm²) in 24 well plates (Orr et al., 2021 (link)) while 3D enteroids were grown as outlined above. Chicken 2D and 3D enteroids were treated with WT or ΔprgH STm, LPS or media alone as outlined above. At 4 and 8 h post-treatment, cells were gently washed with PBS and fixed with 4% w/v paraformaldehyde (TFS) for 15 min at room temperature and blocked with 5% v/v goat serum in permeabilization buffer (0.5% w/v bovine serum albumin and 0.1% w/v Saponin in PBS; Sigma-Aldrich). Permeabilization buffer was used to dilute all antibodies. Cells were stained with mouse anti-human ZO-1 (Abcam, IgG1, clone A12) overnight at 4°C followed by the secondary antibody, goat anti-mouse IgG1 Alexa Fluor^®594 (TFS) for 2 h on ice. Cells were counterstained with Hoechst 33,258 and Phalloidin Alexa Fluor^®647 (TFS) to stain for nuclei and F-actin, respectively. Slides were mounted using ProLong™ Diamond Antifade medium (TFS). Controls comprising of secondary antibody alone were prepared for each sample. Images and Z-stacks were captured using an inverted LSM880 (Zeiss) with 40X and 63X oil lenses using ZEN 2012 (Black Edition) software and were analyzed using ZEN 2012 (Blue Edition). Z-stack modeling was performed using IMARIS software (V9).

To determine whether TfR1 and PEDV co-localize, IPEC-J2 cells and Vero cells seeded on cover slips in 24-well tissue culture plates and infected with PEDV for 1 h at 4°C, rinsed, then incubated at 37°C. At various times post infection cells were fixed in 4% paraformaldehyde for 10 min, rinsed, then permeabilized with 0.1% Triton X-100 in PBS for 5 min, rinsed again then blocked with 5% bovine serum albumin. Cells were incubated with primary antibodies (1:100) overnight at 4°C. Cells were then rinsed and incubated with fluorochrome-conjugated secondary antibodies (1:200) for 30 min at room temperature, washed again three times with PBS and incubated with 1 ug/ml DAPI for 5 min. Images were captured using a Zeiss LSM710 confocal microscope (Carl Zeiss, Germany) and analyzed using ZEN 2012 (Blue edition) (Carl Zeiss).

For live-cell fluorescence imaging microscopy, HepG2 cells were seeded on glass-bottom dishes and transfected with plasmid DNA. Twenty four hours post transfection, fresh medium was applied and cells were monitored by confocal microscope. For on-line investigations, representative cells or cell groups were selected and maintained in buffered medium at 37 °C. Concurrently with treatments, live-cell imaging was started at a rate of one picture per minute. Typically, single treatment experiments were finalised after 15 min and combined treatment experiments after 20 min. Microscopic analyses and image acquisitions were done on an LSM 700 confocal microscope (Carl Zeiss Jena GmbH, Jena, Germany), using ZEN 2012 blue edition and ZEN 2011 black edition software (Carl Zeiss Jena GmbH). Data were analysed and graphed using Microsoft Excel and Prism Software (Graph Pad, La Jolla, CA, USA). Statistical analysis was done using two-way ANOVA and either Dunnett’s or Sidak’s multiple comparisons test. α = 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.

The formation of the SA outer layer was confirmed by confocal laser scanning microscopy (CLSM; LSM 700, Carl Zeiss, Ulm, Germany), using the software ZEN 2012 (Blue Edition, Carl Zeiss, Ulm, Germany). 5(6)-carboxyfluorescein (0.5 mL, 25 mM) was incorporated in the outer infeed of SA. The microparticles were placed onto a microscope slide, covered with a cover glass and subjected to the excitation wavelength of 5(6)-carboxyfluorescein (490 nm), while the fluorescent signal was collected at 520 nm.