Permafluor

Muscle samples were cut in a cryostat in 10 μm serial cross-sections, mounted on glass slides, and stained with previously characterized antibodies to demonstrate muscle fiber types and visualize the muscle cells' basement membrane. In brief, the sections were then incubated for 1 hour at room temperature with primary mouse IgM anti-MyHC I (A4.840, Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa, Iowa City, IA, United States), includeed mouse IgG anti-MyHC IIA (SC-71, DSHB, and rabbit IgG anti-laminin (Z0097, Dako). Muscle sections were then washed in phosphate-buffered saline (PBS) and mounted using PermaFluor (Fisher Scientific). For visualization of capillaries, muscle cross-sections were fixed for 15 min in cold methanol at -20°C and rehydrated in PBS for 10 min at room temperature. A steamer achieved antigen retrieval using a citrate buffer (pH 6.0) for 20 min. Following quenching and protein blocking steps provided by the secondary antibody kit, slides were incubated with primary anti-CD31 antibody (ab9498) or IgG1 isotype control antibody (ab91353) from Abcam (Cambridge, United Kingdom) diluted 1:200 in PBS with 1% BSA overnight at 4°C. After three washes with PBS, secondary antibody incubation and HRP revelation were performed using the HRP/DAB Detection kit (ab64264; Abcam, Cambridge, United Kingdom) following the manufacturer’s instructions.

Mice were euthanized with CO₂ and transcardially perfused with 20 mL Dulbecco's Balanced Salt Solution (DPBS, Thermo Scientific #14190144) followed by 20 mL 4% (wt/vol) paraformaldehyde in DPBS to fix the tissue. Brains were isolated, snap frozen in -40 to -60 °C cold isopentane and stored at − 80 °C. Brains were transferred to − 20 °C 2 h before 8 µm thick coronal sections were obtained. Slices from CTRL, cKO and BK^−/− were collected on the same glass slides (epredia Superfrost™ Plus Adhesion Microscope Slides #J1800AMNZ) together with additional sections from BK^+/+ that served as positive controls. Blocking buffer (BB) consisting of DPBS supplemented with 2% Glycerol, 5% NGS, 0.3% Triton-X-100, 2% (wt/vol) BSA and 50 mM NH₄Cl was used for permeabilization and blocking of unspecific antibody binding. Primary antibodies against BK were diluted 1:1000 in BB and incubated overnight at 4 °C. Sections were then washed thrice with 0.01% Triton-X-100 in DPBS and blocked again with BB for 1 h before incubation with secondary antibody (1:2500) and Hoechst 33342 (1:1000) for 2 h. After three washes with 0.01% Triton-X-100 in DPBS, DPBS and water, sections were mounted with Permafluor (Fisher Scientific, #TA-030-FM). Primary antibodies are listed in Table S7.

Fixed myofibers were permeabilized (0.1% Triton X-100, 0.1 M Glycine in PBS) for 10 min and blocked (5% horse serum, 2% BSA, 0.1% Triton X-100 in PBS) for 2 h. Primary antibodies (listed in Supplementary Table 2) were diluted in blocking buffer and added overnight at 4 °C. Myofibers were washed with PBS and incubated with Alexa Fluor-conjugated secondary antibodies (Life Technologies) for 1 h at room temperature. Myofibers were washed with PBS, stained with DAPI for 5 min, washed again with PBS and mounted onto glass slides using PermaFluor (Fisher).

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).

Animals were anaesthetized with sodium pentobarbitone (Eutasil, Lisbon, Portugal) and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde. Brains were removed and sectioned coronally at a thickness of 50 μm, on a vibrating microtome (VT1000S, Leica, Germany). Sections were incubated overnight, with the primary antibody goat anti-GFP (1:500, Abcam, Cambridge, UK), followed by secondary fluorescent antibody (1:1000, Invitrogen, MA, USA). All sections were stained with 4’,6-diamidino-2-phenylindole (DAPI; 1 mg ml-1) and mounted using mounting media (Permafluor, Invitrogen, MA, USA).