Dapi counterstain

Anti-nuclear immunofluorescent staining was performed as previously described (23 (link)). Briefly, Kallestad HEp-2 slides (Bio-Rad) were stained overnight with 1 μL of serum from either Lyn^–/–IgD^+/– or wild-type mice. After washing, antibody binding was detected using an anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific) and DAPI counterstain (Thermo Fisher Scientific). Cells were imaged with the Crest LFOV Spinning Disk/C2 confocal microscope at ×1000 magnification under identical camera exposure and laser settings for knockout and control mice. Micrograph exposure was normalized to secondary-only negative control in FIJI and applied to all images at once.

The cells treated with Ps-GOS were grown on sterile collagen-coated coverslips in 24 well plates. In brief, coverslips were fixed with 4% paraformaldehyde 20 min, quenched with 50 mM NH₄Cl for 2 × 10 min, washed with PBS, and permeabilized with 0.1% Triton X-100 for 10 min at RT. After blocking with 0.5% FSG in PBS for 20 min, each coverslip was incubated overnight at 4 °C with polyclonal rabbit antimouse BMP-2 (Abcam, Milton, UK), followed by removing primary antibody and washing 3 × 5 min in blocking solution before incubation for 1 h with FITC-conjugated secondary antirabbit IgG (Alexa Fluor 680) (Thermo Fisher Scientific, Waltham, MA, USA) and DAPI counterstain (Thermo Fisher Scientific, Waltham, MA, USA). Coverslips were washed 3 × 5 min in blocking solution and mounted on to slides using Prolong^TM Gold antifade reagent (Invitrogen, Carlsbad, CA, USA). Fluorescence images were acquired with Nikon Eclipse Ti fluorescence microscope.

For mouse tissue, additional perilesional tissue from previous passive transfer experiments and optimizations were used to minimize animal numbers. Hematoxylin and eosin stains were performed using standard protocols. For both human and mouse, FFPE blocks were sectioned routinely and stained with the following antibodies: CLE4D (MBS9607710, rabbit polyclonal, Mybiosource, San Diego, CA, USA), CLEC4E (BS08541R, rabbit polyclonal, Bioss, Woburn, Massachusetts, USA), and CLEC4N (clone IMG3D1, ab107572, mouse IgG3 monoclonal, Abcam, Boston, Massachusetts, USA). Human neutrophils were stained for MPO (AF3667, goat polyclonal, R&D Systems, Minneapolis, Minnesota, USA), while mouse samples were stained for Ly6G (clone RB6-8C5, SC-53515, rat monoclonal IgG2b, Santa Cruz, Dallas, Texas). Secondary antibodies and DAPI counterstain were purchased from ThermoFisher (Rockford, IL, USA). Mouse direct immunofluorescence was performed as previously described, using anti-rabbit secondary antibody to confirm successful passive transfer (17 (link)). All slides were immediately photographed following staining using an Evos FL microscope (ThermoFisher, Rockford, IL, USA). The frequency of dual-positive cells was quantified in ImageJ v1.52 (Bethesda, Maryland, USA)

HL-60 neutrophils were incubated with P. gingivalis in 96-well plates for 12 h at 37°C. Neutrophils were fixed in 3.7% formaldehyde for 10 min, permeabilized in 0.5% Triton X-100 for 5 min, blocked in 2% BSA, and stained with primary antibody and appropriate isotype-matched negative control antibody. This was followed by incubation with biotinylated secondary antibody and ABC reagent and visualized by incubation with Alexa Fluor 546-conjugated streptavidin (#S11225, Thermo Fisher Scientific) with DAPI counterstain (#62248, Thermo Fisher Scientific). Images were captured at a magnification of 200× with a fluorescence microscope (Nikon) with the same exposure time for experimental and negative control groups. The capture time was set so that control antibody images were negative. Image analysis was performed using NIS Elements AR image analysis software. The percentage of immunofluorescence positive cells or mean fluorescence intensity (MFI) was measured.

To visualize early and late gene expression, ADSCs, RK13 and three pancreatic adenocarcinoma cell lines (murine Pan02, human Panc-1 and AsPC-1) were infected with vMyx-EGFP/tdTr (MOI = 5). Cells were plated into 4-well chamber slides (5 × 10⁴ cells/well). After 24 h p.i., cells were washed with PBS⁻ and fixed in paraformaldehyde (4%) for 10 min at room temperature (RT). Cell nuclei were stained with DAPI Counterstain (Life Technologies™, ThermoFisher Scientific, Warsaw, Poland). Infection was evaluated using fluorescence microscopy (Zeiss LSM 710 confocal workstation).

The cells were adhered to 10-well slides, fixed, and permeabilized as previously described (21 (link)). The cells were blocked with Image-iT FX Signal Enhancer (Life Technologies, Grand Island, NY, USA) for 15 min at room temperature, and then either singly or doubly stained with primary antibodies. Subsequent labeling with Alexa Fluor-conjugated secondary antibodies and DAPI counterstain (Life Technologies) were performed to visualize primary antibodies and nuclei, respectively. Stained cells were viewed using the Olympus FV10i fluorescence confocal microscope. Images were analyzed using FluoView software (Olympus, Melville, NY, USA).