Kul01

Cells isolated from the spleen, liver, and intestinal epithelium were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (L3457; Thermo Fisher, USA) and the following fluorescent antibodies to chicken CD3 (CT-3, Southern Biotech, USA), CD4 (CT-4, Southern Biotech, USA), CD8α (CT-8, Southern Biotech, USA), CD44 (AV6, Southern Biotech, USA), TCRγδ (TCR-1, Southern Biotech, USA), monocytes/macrophages (KUL01, Southern Biotech, USA), MHCII (2G11, Southern Biotech, USA), Bu-1 (AV20, Southern Biotech, USA), and CD45 (LT-40, Southern Biotech, USA). Stained cells were analyzed using a CytoFLEX flow cytometry and the data were analyzed using CytExpert Software (both from Beckman Coulter, USA).

Jejunal cryosections, 8 μm thick, were stained with specific antibodies using an indirect immunoperoxidase staining method as described by Schokker et al. [29 (link)]. Briefly, slides were treated for endogenous peroxidase activity, blocked with BSA, and incubated with monoclonal antibodies against CD4⁺ cells, CD8⁺ cells, or macrophage-like cells (CT-4, 1:200; CT-8, 1:200; and KUL01, 1:50, respectively; Southern Biotech, Birmingham, AL), followed by peroxidase-conjugated rabbit anti-mouse Ig (P0161, Dako, Denmark). Peroxidase activity was detected by 3,3-diaminobenzidine, and sections were counterstained with haematoxylin. Negative controls were performed by omitting of the primary antibody. For each sample 3 to 4 mm² mucosa (without muscular layers) were evaluated by 10x magnification on a bright field microscope. Subsequently, the samples were further analysed using Olympus cellSens Dimension (version 1.7.1) software. First positive-stained cells were counted, secondly these cells were averaged per time point and group, and lastly they were represented as positive-cells per tissue area (square mm). A Student’s T-test was performed to calculate the significance between the treatment and control on each time-point separately.

Immunofluorescent Assay (IFA) to detect macrophages (KUL01+), CD4+ T cells, and CD8α+ T cells was performed as described previously [24 (link)]. In brief, the OCT preserved lung tissue sections were fixed in cold acetone for 5 min (min) and blocked in 5% goat serum diluted in a Trisma buffered saline (TBS) buffer at room temperature for 30 min. The primary antibodies, the mouse monoclonal antibody specific for chicken macrophages/monocytes, KUL01+ (Southern Biotech, Birmingham, AL, USA), CD8α (CT-8, Southern Biotech, Birmingham, AL, USA), were used in a 1:200 dilution in a 5% goat serum for 30 min. Then, the secondary antibody, goat anti-mouse IgG (H + L) conjugated with Dylight^® 550 (Red fluorescence, Bethyl Laboratories Inc., Montgomery, TX, USA) was used for 1 h. For CD4+ staining, chicken CD4 antibody (CT-4, Southern Biotech, Birmingham, AL, USA) was used as the primary antibody followed by biotinylated goat anti-mouse IgG (H + L) (Southern Biotech, Birmingham, AL, USA) used in a 1:250 dilution. Dylight^® 488 (green fluorescence) streptavidin was used as a final step followed by mounting the slides with Vectashield^® mounting medium with DAPI (Vector Laboratories Inc., Burlingham, CA, USA).