sEVs were incubated with the fluorescent dye DiD (1,1′-dioctadecyl-3,3,3′,3′- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (Thermo Fisher, Grand Island, NY, USA)), as prescribed previously [31 (link)]. In short, sEVs were incubated with 1 µM DiD at 37 °C for 30 min. The sample was centrifuged for 70 min at 100,000× g using an Optima XPN ultracentrifuge (Beckman Coulter, Pasadena, CA, USA) in the 70Ti rotor (k = 156) to remove the excess dye. The resulting pellet was resuspended in PBS (Ca/Mg free). A total of 2 billion labelled sEVs in 2 doses of 3 µL per nostril were administered intranasally after pretreatment with hyaluronidase as described above.
Mice were sacrificed by CO2 exposure and perfused with ice cold PBS supplemented with 5 U/mL sodium heparin, and brains were collected and imaged in an IVIS Lumina (Perkin Elmer, Waltham, MA, USA) using the 640/700 nm excitation/emission filters. Intact brains were imaged from the axial/horizontal plane. Coronal images were obtained on 1.0 mm slices prepared using the mouse slicing matrix (Zivic catalog #BSMAS001-1) across hippocampal regions corresponding to section 19 of the C57BL/6J Atlas (−1.28 mm from bregma) (http://www.mbl.org/atlas170/atlas170_frame.html , accessed on 5 April 2021).
Mice were sacrificed by CO2 exposure and perfused with ice cold PBS supplemented with 5 U/mL sodium heparin, and brains were collected and imaged in an IVIS Lumina (Perkin Elmer, Waltham, MA, USA) using the 640/700 nm excitation/emission filters. Intact brains were imaged from the axial/horizontal plane. Coronal images were obtained on 1.0 mm slices prepared using the mouse slicing matrix (Zivic catalog #BSMAS001-1) across hippocampal regions corresponding to section 19 of the C57BL/6J Atlas (−1.28 mm from bregma) (
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