Immunofluorescent Staining of Chicken Enteroids

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).