Thunder fluorescent microscope

This method was previously described.^{9 (link)} In brief, for teased nerve fibre staining, sciatic nerves were fixed in 4% paraformaldehyde (PFA) overnight and teased into individual fibres on glass slides. For paraffin nerve staining, sciatic nerves were fixed, embedded in paraffin and sectioned into 5 μm-thick slices. The slides were incubated with primary antibodies at 4°C overnight. Following a wash, the slides were incubated with secondary antibodies for 1 h. The stained slides were observed under a Leica THUNDER fluorescent microscope.

For in vitro EV uptake experiments, HMEC-1 (1.4 × 10⁴/well) were seeded on cover glass in a 24-wells plate and incubated for 24 h (647-NHS ester-labeled) PalmtdTomato- or PalmGFP-EVs were administered to the culture media of the recipient cells and incubated for 4 h at 37°C, after which HMEC-1 were washed with PBS and fixed using 4% paraformaldehyde (PFA). Cells were stained with Alexa Fluor 555 or -488 phalloidin (Thermo Fisher Scientific), and nuclei were stained using 1:10,000 Hoechst 33342 (Invitrogen) for 30 min at RT. The cover glass was mounted on a slide using Fluoromount-G (SouthernBiotech) or Mowiol and fluorescent images were taken using a Thunder fluorescent microscope (Leica).