Fluoview fv1000 mpe microscope

Three different HUVEC/DPSC co-culture systems were established: (1) EV-loaded gels containing co-cultured cells cultivated in EBM-2; (2) gels containing co-cultured cells but no EVs cultivated in EBM-2; (3) gels containing co-cultured cells but no EVs cultivated in an 1:1 mixture of EBM-2 and CM. The two cell types were prepared individually at concentrations of 6 × 10⁶ cells/mL, mixed at a 1:1 ratio and applied to the gels at a concentration of 1.2 × 10⁶ mixed cells per 200 μL of fibrin gel. The medium (EBM-2 or CM/EBM-2) was supplemented with 0.16 mg/mL tranexamic acid (Pfizer, New York, NY, USA). Samples were analyzed by TPLSM on days 5, 7 and 10 to visualize vascular and collagen structures using an Olympus FluoView FV1000 MPE microscope (Olympus, Hamburg, Germany). After being fixed in 4% paraformaldehyde for 2 h, the hydrogel samples were stained with primary and secondary antibodies. Information about the antibodies is listed in Supplementary Table S3. Each sample was imaged at random locations with a stack depth of 80 μm using a 25× magnification water immersion lens (Olympus XLPLN 25 × WMP-SP, NA 1.05, Hamburg, Germany). The dimensions of the 3D stacks were set to 500 × 500 × 80 μm. Imaris imaging software (Bitplane, Zurich, Switzerland) was used to analyze the 3D stacks.

Immunofluorescence staining was performed following a previously described protocol [12 (link), 14 (link)]. In brief, brain tissue slides were dried overnight, washed twice with PBS (0.01 M) for 10 min, and blocked with blocking serum (2% normal goat serum and 0.3% Triton X-100 in PBS) for 1 hour. The slides were then incubated with the aforementioned primary antibodies (Santa Cruz Biotechnology) overnight at 4°C. The next day, the slides were treated with secondary TRITC/FITC-labelled antibodies (1,100) (Santa Cruz Biotechnology) for 2 h. Slides were washed with PBS (twice for 5 min) and mounted with 4′,6′-diamidino-2-phenylindole (DAPI) and Prolong Antifade Reagent (Molecular Probe, Eugene, OR, USA). Finally, the stained slides were examined under a confocal laser-scanning FluoView FV 1000 MPE microscope (Olympus, Tokyo, Japan).