Cm 3000

Optimized MENDs encapsulating Cy5-labeled siRNA were administered into male ICR mice (5–6 weeks old). Five min before projected sacrifice, FITC-conjugated isolectin B4 (40 μg/mouse) was intravenously injected via the tail vein to stain blood vessels. At 30 min after intravenously injection of optimized MEND, each animal was perfused with PBS to remove blood from the liver, then with 4% paraformaldehyde (PFA)-PBS for fixation. Liver tissues were excised and further fixed with 4% PFA-PBS for 24 hr at 4°C, then submerged in 20% sucrose-PBS for 4 hr at 4°C. The liver was embedded in OCT compound (Sakura Fine Technical, Tokyo, Japan) and snap-frozen in liquid nitrogen. Frozen samples were cut in 30 μm-thick sections (LEICA CM3000, Leica Microsystems, Wetzlar, Germany). The samples were stained with Hoechst33342 to detect nuclear DNA, and observed at an excitation wavelength of 633 nm using a laser-equipped Nikon A1 (Nikon Co. Ltd., Tokyo, Japan) with a x60 objective lens.

The A. niger N402 wild type cells were harvested from solid MM, covered with a porous cellophane membrane, embedded in optimal cutting temperature compound (OCT; Tissue Tek, Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands) and frozen in a cryostat Leica CM3000, (Leica Microsystems GmbH, Wetzlar, Germany) at −30 °C and cut into 10 µm-thick cryosections. The sections were fixed with 2% paraformaldehyde for 20 min at 22 °C. After washing with PBS (pH 7.4) at 22 °C the cells were blocked with 1 % BSA in PBS. The sections were incubated with anti-mouse NigA2 antibodies (1:1000 in 1% BSA in PBS) [34 (link)] for 2 h at 37 °C and then washed with PBS. After washing, the sections were incubated overnight with secondary goat anti-mouse-Alexa-488 antibodies (1:400) (Molecular Probes, Thermo Fisher Scientific Inc., Waltham, MA, USA) in 1% BSA in PBS, at 4 °C. The next day, the sections were washed with PBS and mounted in Vectashield for nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). Immunostained embedded cryoresin slides were observed with the inverted confocal microscope as described above.

The fixed jejunal samples were immersed in 30% sucrose in 50 mM PBS for two days at 4 °C, embedded in Tissue-Tek OCT compound (Sakura FineTechnical, Tokyo, Japan), and frozen on dry ice. Cryosections (thickness: 4 μm) were obtained using a cryostat (CM3000; Leica Microsystems, Wetzlar, Germany) and blocked with 2% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 30 min at 30 °C. Tissues were incubated in antibody retrieval buffer with 0.01 M citrate, pH 6.0, for 10 min at 90 °C, and then with primary antibodies against the TJ proteins claudin-1 (Cat#717800), claudin-2 (Cat#51600), claudin-7 (Cat#349100), and zonula occludens (ZO-1) (Cat#617300) [Thermo Fisher Scientific (Waltham, MA, USA)], claudin-3 (SAB4200607), and claudin-5 (SAB4200537) [Sigma-Aldrich (St. Louis, MO, USA)] at 4 °C overnight. After being washed with PBS, the fixed jejunal tissues were incubated with the secondary antibody Alexa-Fluor-488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 30 °C. Nuclei were visualized in Vectashield Hardset Mounting medium with DAPI (Vector Laboratories, CA, USA). Imaging was performed using a confocal laser microscope, FluoViewTM FV1000 (Olympus, Tokyo, Japan), equipped with ×20 and ×40 objective lenses.