Zen 2012 black edition software

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).

Immunofluorescence of fixed whole 96-Transwell inserts was performed as described above. Six randomly selected areas per insert were captured using a 20× Plan-Apochromat objective on a LSM 710 confocal microscope system (Zeiss, Oberkochen, Germany) with the following settings: Alexa Fluor 488 channel, 5% laser power, master gain 600, acquisition speed 9. To account for unevenness in specimen surface and thickness, Z-stack imaging of 25 vertical stacks per area was applied and maximum intensity projection was performed using the ZEN 2012 Black Edition software (Zeiss, Oberkochen, Germany). Image analysis was performed using the Fiji for ImageJ software (11). Each image was first converted to 8-bit format and then converted to binary at a threshold of 20–255. To avoid overlapping of structures, the watershed function was performed to accurately cut structures apart. Afterwards, nuclei/ surfactant positive areas were analyzed by using the 3D Objects counter tool in ImageJ. For Nuclei counting areas within 60 px were analyzed and summarized per image. Surfactant positive cells were counted based on > 100 px. For each analyzed Transwell insert, SFTPC positive cell counts were normalized to total cell number (based on nuclei counting) and the percentage of SFTPC positive cells in hiPSC-derived AT2-like cells at day 27 were calculated from the six selected areas.