Optical microscope

We obtained consecutive (serial) cuts of 3-μm in the paraffined tissue block for the immunohistochemical analysis. The antigens were retrieved after deparaffinisation and rehydration in ethanol solutions. Afterwards, we blocked endogenous peroxidase activity with 0.1% H2O2 for 5 min at room temperature. The slides were incubated separately with primary antibodies against CD1a (Dako, Carpinteria, CA, USA), CD83, CD123, Factor XIIIa, CD163 (Leica Biosystems, Wetzlar, Germany), CD207 (Monosan, Uden, The Netherlands), CD209, CD68 (DakoCytomation, Glostrup, Denmark), and CD208 (Dendritics, Lyon, France) followed by the LSAB method (Dako, Carpinteria, CA, USA) and the reactions were developed by incubating the sections with 0.6 mg/ml 3,3’diaminobenzidine tetrahydrochloride (Sigma-Aldrich, Saint Louis, MO, USA). The slides were counterstained with Carazzi’s hematoxylin. Negative controls were obtained by omitting primary antibodies. The results were observed under an optical microscope with 200× magnification (Leica Biosystems, Wetzlar, Germany). Ten consecutive and representative fields of the intraepithelial and subepithelial areas were selected for photomicrography (Leica Application Suite–LAS). Two independent examiners counted positive cells using the Image J software (version 1.52, NIH, Bethesda, Rockville, MD, USA).

The P. xiamenensis overnight-grown culture was inoculated into 10 mL of fresh marine broth at 1% (v/v) inoculation. Cultures were incubated at 30 °C with vigorous shaking. When the OD₅₉₅ reached 0.4 (1.6 × 10⁸ CFU/mL), the cells were collected by centrifugation at 2657× g at 4 °C for 10 min. The supernatant was discarded and the pellet was collected and re-suspended in 1 mL of PBS and placed on ice. Cell numbers were adjusted to 0.2, 0.4, 0.6, and 0.8 OD₅₉₅, to bring cell numbers to 7.6 × 10⁷, 1.6 × 10⁸, 2.6 × 10⁸, and 3.8 × 10⁸ CFU/mL, respectively. Zebrafish at 60 hpf were collected into 6-well plates containing egg water, and co-inoculated with P. xiamenensis by adding 200 μL of each inoculation dose in replicates. Fish larvae without bacterial inoculations were test controls. Plates containing fish larvae and bacteria were incubated at 28 °C. Larval mortality was monitored for 96 h. The morphological changes were observed under an optical microscope (Leica Biosystems, Wetzlar, Germany). The average heart rate was also evaluated for the control and treatment groups under microscopic observation.

A549 (7 × 10⁴/well) or Capan-I (1 × 10⁵/well) cells, either wild-type or MET^−/−, were resuspended in cell culture medium with 1% FBS and seeded in the upper compartment of transwell chambers (8.0 μm pore polycarbonate membrane insert; Corning Inc., New York, NY, USA) pre-coated with 40 μg/well of Matrigel Reduced Growth Factors (Corning Inc.). The lower compartment of the chamber was filled with 1% FBS cell culture medium supplemented with HGF (25 ng/mL). For A549, either wild-type, MET^−/− or MET^−/− back-expressing MET upon lentiviral vector transduction, the following conditions have also been tested: lower chamber medium + 5% FCS + HGF (25 ng/mL); upper chamber medium + 1% FCS + HGF (25 ng/mL). After 24 h, cells on the upper side of the transwell filters were mechanically removed, while cells migrated through the membrane were fixed with 11% glutaraldehyde and stained with 0.1% crystal violet. All images were captured with an optical microscope (Leica Biosystems). Images were quantified with Image-J v1.59s software.

A Renishaw InVia spectrometer, working in a confocal mode, connected to a Leica optical microscope with a 100× magnification objective (NA = 0.9) and an argon laser line of 514.5 nm were used to measure single erythrocytes. The laser power was kept low, ca. 1–3 mW at the sample, to ensure minimum invasion into the cells. The averaged spectra were made in Thermo Scientific Omnic v. 9.3 software.

For alkaline phosphatase staining, on day seven, the cells were washed with PBS, fixed in 3% glutaraldehyde for 30 min, and then stained with BCIP/NBT ALP kit (LEAGENE, Beijing, China). The images were acquired with an optical microscope (Leica, Germany).