Jem 2000exii microscope

For TEM, exosomes were fixed in 2% paraformaldehyde (Sigma-Aldrich) and spread onto carbon/Formvar-coated grid (Ted Pella, Inc.) for 30 min at room temperature. The grid was then washed with DPBS and fixed with 1% glutaraldehyde (Sigma-Aldrich). TEM images were captured with a JEOL JEM-2000EXII microscope (JEOL, LTD). For NTA, exosomes were diluted to 10⁶ to 10⁹ particles/ml to optimize particle concentration in a view of field for analysis of exosome size distribution using a NanoSight instrument (NS300, Malvern) equipped with a CMOS camera and a 488-nm blue laser.

Crawling 3^rd instar larvae of the appropriate genotypes were dissected in gluteraldehyde fix solution (2% paraformaldehyde, 2% gluteraldehyde, 0.2 M of cacodylate and 0.005% CaCl₂). Fixed larvae were then processed, embedded and sectioned (70 nm) by the UTHSC Neuroscience Institute Imaging Core. Transmission electron microscopy images were captured on a JEOL JEM-2000EXII microscope at 60 kV to visualize the active zones within NMJ boutons. Images containing boutons with clearly identifiable active zones were used for statistical analysis. A rectangular box extending 250 nm inward from the AZ and running the entire length of each AZ was used for analysis. Bouton volumes were measured using ImageJ. Statistical analysis via One-way ANOVA was performed using Prism 5.0 software (GraphPad Software, San Diego, CA, USA).