Virtual slide system

After completing the experiment, mice were sacrificed and paws were fixed in 10% neutral-buffered formalin and decalcified with 10% EDTA, pH 7.4. The paws were then embedded in paraffin and cut into 5-μm thick sections. The deparaffinized sections were stained with hematoxylin and eosin and observed under light microscope. For immunostaining, the sections were incubated for 1 h at room temperature (RT) with monoclonal antibody (mAb) to mouse YL-6G/-6C neutrophil marker (1 : 50, Hycult Biotech, Uden, The Netherlands) or mAb to rat anti-mouse F4/80 antigen (1 : 100, AbD Serotec, Co., Oxford, UK). Goat anti-rat IgG2b conjugated with horseradish peroxidase (HRP, 1 : 250, AbD Serotec, Co.) was used, and immunoreactive cells were visualized by staining with 3,3-diaminobenzidine solution. Images were observed using virtual slide system (Olympus, Co., Tokyo, Japan).

EVs derived from CCM+FBS, SFM1, and SFM2 were labeled with PKH26 (Sigma, St. Louis, MO), as described previously (28 (link)). BV-2 cells were cultured in DMEM with 5% heat-inactivated FBS and 1% antibiotic-antimycotic solution at 37°C in 5% CO₂. After BV-2 cells reached about 70–80% confluency, they were detached using 0.25% trypsin-EDTA solution and seeded at 2.5 × 10⁵ cells per chamber in chamber slides (Iwaki, Tokyo, Japan) overnight. The labeled EVs were added and co-cultured for 6 h. The nuclei of BV-2 cells were stained with 4′,6-diamidino-2′-phenylindole-dihydrochloride (DAPI) (Invitrogen, Carlsbad, CA). Fluorescence microscope images were acquired using a Virtual Slide System (Olympus Corporation, Tokyo, Japan).