Fluoviewtmfv3000

During the EVs isolation process, and before qEV column use, mouse brain EVs were labeled using 5 µM DiI (cat. No. D282, Invitrogen/Molecular Probes) for 30 min at 4 °C (gentle rotation). Then, the isolation protocol was concluded to obtain a pellet of DiI-labelled sEVs. DiI-labeled EVs were quantified by NTA and BCA as described above. Animals were stereotaxically injected with DiI-labeled EVs and corresponding controls (see below) into the outer molecular layer (OML) of the dentate gyrus (DG) with the following brain coordinates: anteroposterior, − 2.18 mm; lateral, 1.25; dorsoventral, − 1.60. The different control groups included injection of equal volumes of PBS, DiI dye alone, or DiI dye alone which was processed through the release protocol. Animals were sacrificed 1 day and 4 weeks after the injection as previously described [28 (link)]. Briefly, deeply anesthetized animals were transcardially perfused with saline and PFA (4%). After post-fixation for 24 h at RT in 4% PFA, brains were placed in 30% sucrose and sectioned using vibrating-blade microtome (VT1000S, Leica, Germany). 30 µm slices were incubated for 10 min, room temperature, with DAPI (1 mg/ml) and mounted using mounting media (Permafluor, Invitrogen, MA, USA). Images were collected by confocal microscopy (Olympus FluoViewTMFV3000, Olympus, Tokyo, Japan).

A549 cells were grown on 6-well plate containing 12 mm cover slips to 80–90 % confluency. Cells were infected with influenza A virus at an MOI of 2 at 1 h time point and then processed for IFA. After 24 h post infection, cells were washed twice with PBS and then fixed with chilled acetone at − 20 °C for 20 min. Acetone was removed and fixed cells were washed with PBS and subjected to IFA staining using primary antibody against viral M1 and incubated overnight at 4 °C in moist chamber. Next day, coverslips were washed with chilled PBS thrice and then probed with anti-rabbit FITC (green) conjugated secondary antibody (Sigma-Aldrich) at 37 °C for 1 h. After three washes nuclei were stained with 2.5 µg/ml of Hoechst stain for 10 min at RT. Cover slips were mounted on glass slides and the protein expression was visualized under Confocal Laser Scanning Microscope (Olympus FluoviewTM – FV3000) equipped with HeNe laser (488 nm), HeNe laser (640 nm) and pulse diode laser (405 nm). Images were acquired with GaAsP 100X NA: 1.45 oil immersion objective using CELLSENS 3.2 software.