Observer d1 inverted microscope

Human umbilical cords were obtained under sterile conditions after birth from full-term infants, with the consent of the parents. The umbilical cord blood vessels (two arteries and one vein) were removed and the remaining tissue was diced into small fragments (1 mm³ pieces) and treated with 2 mg/mL collagenase II for 3–4 h at 37 °C. Cell suspension was centrifuged, washed and resuspended in α-minimal essential medium (GIBCO, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (GIBCO, Gaithersburg, MD, USA) and 10 ng/mL basic fibroblast growth factor (bFGF) (Sigma, St. Louis, MO, USA). The cells were plated at a density of 1 × 10⁴ cells/cm² and cultured at 37 °C in a humidified atmosphere containing 5% CO₂. The medium was changed every 3 days and passaged by trypsinization when the cells reached 70–80% confluence. Cells at the passage 14 of were used in our experiments. 2 × 10⁵ cells were seeded on the functionalized slides distributed in 6 wells plate. 24 h later, the cells were fixed in ethanol 70% and stained with 50 µg/mL propidium iodide. The morphology of lived or stained cells were observed using Observer.D1 inverted microscope (Carl Zeiss AG, Dublin, CA, USA).

A Zeiss Observer D1 inverted microscope using Zeiss ZEN software was used to capture the images of the live zebrafish embryos.
Wild-type zebrafish were maintained at 28.5 °C in a 10 h dark and 14 h light period in a circulating water system provided by Pentair (www.pentair.com). The fish were kept following the national and international guidelines for the care and use of laboratory animals. The fertilized eggs were obtained by the natural pairwise breeding of adult fish. The approval of the Ethics Committee for the use and care of laboratory animals was not needed, as zebrafish larvae less than 5 days post fertilization were used [30 (link)].
A stock solution of 50 mM was prepared by dissolving the compounds in dimethyl sulfoxide (DMSO). At least 30 fertilized eggs were sorted and placed in 60-mm sterile glass Petri dishes. A serial dilution of the compounds was prepared by adding the required amount of the compound directly into 5 mL water, and the zebrafish embryos were then exposed to serial dilutions (0.5 to 1000 µM) of each compound overnight. The number of dead and living embryos were counted after the overnight exposure.

Caco-2 cells (5 × 10⁵ cells/well) were cultured and treated for 48 h with the control medium, medium with LPS (1 μg/mL), and medium with LPS (1 μg/mL) and BWI mix (10⁷ CFU/mL). After being washed with PBS, Caco-2 cells were fixed with 4% paraformaldehyde in PBS pH 7.4 for 10 min and permeabilized with PBS containing 0.25% Triton X-100 for 10 min at 25 °C. Next, Caco-2 cells were incubated with 1% BSA in PBS for 30 min at room temperature. The primary antibody occludin (Cell Signaling, Danvers, MA, USA, cat. no. #5506) was diluted 1:20 and incubated for 1 h overnight at 4 °C and a secondary antibody (fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich, St. Louis, MO, USA, cat. no. F9887) was diluted to 1:100 and incubated for 30 min at room temperature. Finally, 300 nM DAPI solution (Invitrogen, Waltham, MA, USA, cat. no. D1306) was added to Caco-2 cells and incubated for 10 min at room temperature for nuclear counterstaining. After mounting with a mounting solution (Invitrogen, Waltham, MA, USA, cat. no. 00-4958-02), images were acquired using an Observer D1 inverted microscope (Carl Zeiss, Jena, Thuringia, Germany) equipped with Automatic Component Recognition module and AxioVision 3.1 software (Carl Zeiss, Jena, Thuringia, Germany) using a 40×/1.0 objective lens.